Increasing levels of nicotinic alkaloids in plants

ABSTRACT

Four genes, A622, NBB1, PMT, and QPT, can be influenced for increasing nicotinic alkaloid levels in  Nicotiana  plants, as well as for synthesizing nicotinic alkaloids in non-nicotine producing plants and cells. In particular, overexpressing one or more of A622, NBB1, PMT, and QPT may be used to increase nicotine and nicotinic alkaloid levels in tobacco plants. Non-nicotine producing cells can be engineered to produce nicotine and related compounds by overexpressing A622 and NBB1.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.11/520,036, filed Sep. 13, 2006. The content of this application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology andregulation of nicotinic alkaloid synthesis. Thus, the invention relates,inter alia, to methodology and constructs for increasing the level ofnicotinic alkaloids in a Nicotiana plant, and to cells that aregenetically engineered to produce nicotinic alkaloids and relatedcompounds, when they would not do so otherwise.

BACKGROUND OF THE INVENTION

Presently, several nicotine biosynthesis enzymes are known. For example,the tobacco quinolate phosphoribosyl transferase (QPT) gene has beencloned, see U.S. Pat. No. 6,423,520 and Sinclair et al., Plant Mol.Biol. 44: 603-17 (2000), and its suppression provides significantnicotine reductions in transgenic tobacco plants. Xie et al., RecentAdvances in Tobacco Science 30: 17-37 (2004). Likewise, suppression ofan endogenous putrescine methyl transferase (PMT) sequence has beenshown to reduce nicotine levels but increase anatabine levels by about2-to-6-fold. Hibi et al., Plant Cell 6: 723-35 (1994); Chintapakorn andHamill, Plant Mol. Biol. 53:87-105 (2003); Steppuhn et al. PLoS Biol2:8:e217:1074-1080 (2004).

While previous research efforts have focused on using nicotinebiosynthesis enzymes for reducing nicotine in plants, very littleresearch has addressed the role of nicotine biosynthesis enzymes inincreasing nicotinic alkaloid synthesis. This lack of up-regulation datamay be attributed to the fact that overexpressing a known nicotinicalkaloid biosynthesis gene, such as PMT, or QPT, will not necessarilyincrease plant production and accumulation of secondary metabolites.That is, it does not necessarily follow that because down-regulating anicotinic alkaloid biosynthesis gene reduces alkaloid production andaccumulation, overexpressing the same nicotinic alkaloid biosynthesisgene will increase nicotinic alkaloid production and accumulation.

Due to the paucity of research, there is a need for identifying genesthat increase nicotine biosynthesis and accumulation. For example,because nicotinic alkaloids play an important role in protecting plantsagainst insects and herbivores, it is likely to be advantageous toincrease nicotinic alkaloid synthesis in a host plant. From an herbivoryperspective, increased nicotine synthesis and accumulation would providean environmentally acceptable means for mediating plant-pestinteractions.

From the cigarette industry's perspective, where nicotine is thephysically and psychologically active component in cigarette smoke, itmay be advantageous to increase nicotine content in tobacco by geneticengineering. Research studies demonstrate that when supplementarynicotine is physically added to cigarette tobacco from an outsidesource, smokers inhale less of the more harmful components of smoke suchas tar and carbon monoxide. See Armitage et al., Psychopharmacology 96:447-53 (1988), Fagerström, Psychopharmacology 77: 164-67 (1982),Russell, Nicotine and Public Health 15: 265-84 (2000), and Woodman etal., European Journal of Respiratory Disease 70: 316-21 (1987).Likewise, a report by The Institute of Medicine of the U.S. on potentialreduced-exposure products (PREPs) concluded that “retaining nicotine atpleasurable or addictive levels while reducing the more toxic componentsof tobacco is another general strategy for harm reduction.” See CLEARINGTHE SMOKE, ASSESSING THE SCIENCE BASE FOR TOBACCO HARM REDUCTION, IOM atpage 29 (2001); commonly referred to as the “IOM Report” by the tobaccoindustry.

In addition to the more traditional applications for increased nicotineproducts, such as cigarettes and other tobacco products, recentpharmacological studies suggest a therapeutic role for nicotine andrelated compounds. For example, several research groups are presentlystudying drugs that target nicotine receptors as a means for treatingcognitive impairments, such as Alzheimer's disease, schizophrenia, andage-related memory loss. Singer, “The Upside to Nicotine,” TechnologyReview (Jul. 28, 2006). Acetylcholine receptor ligands, such asnicotine, have been demonstrated to have effects on attention,cognition, appetite, substance abuse, memory, extra pyramidal function,cardiovascular function, pain, and gastrointestinal motility andfunction. U.S. Pat. No. 5,852,041. Thus, there are therapeutic benefitsof nicotine and related compounds, and thus there is a need for improvedmethods for producing them.

Accordingly, there is a continuing need to identify additional geneswhose expression can be affected to increase nicotinic alkaloid contentin plants, in particular, nicotine in N. tabacum plants, as well asproduce nicotine and related compounds in non-nicotine producing cells.

SUMMARY OF THE INVENTION

Four genes, A622, NBB1, PMT and QPT, can be influenced for increasingnicotinic alkaloid levels in Nicotiana plants, as well as synthesizingnicotinic alkaloids and related compounds in non-nicotine producingcells.

In one aspect, the invention provides a method for increasing nicotinicalkaloids, such as nicotine, in a Nicotiana plant, by overexpressing atleast one of A622 and NBB1 relative to a control plant. In oneembodiment, A622 is overexpressed. In another embodiment, NBB1 isoverexpressed. In another embodiment, A622 and NBB1 are overexpressed.In a further embodiment, A622 and NBB1 are overexpressed and at leastone of QPT and PMT is overexpressed. In a still further embodiment, QPTand A622 are overexpressed.

In another embodiment, an increased nicotine plant, and products therefrom, are produced by any method overexpressing one or more of A622,NBB1, PMT, and QPT. In a further embodiment, products are selected fromthe group consisting of a cigarette, a pharmaceutical, and anutraceutical.

In another aspect, the invention provides a method for producingnicotinic alkaloids, comprising expressing NBB1 and A622 heterologouslyin a plant or cell that otherwise does not produce nicotinic alkaloids.In one embodiment, NBB1 and A622 expression occurs in a cell selectedfrom the group consisting of a bacterial, yeast, filamentous fungi,algae, mammalian, and insect cell.

In another embodiment, an increased nicotinic alkaloid plant is producedby expressing NBB1 and A622 heterologously in a plant or cell thatotherwise does not produce nicotinic alkaloids. In another embodiment, anicotinic alkaloid product is produced by expressing NBB1 and A622heterologously in a plant or cell that otherwise does not producenicotinic alkaloids.

In another aspect, there is provided a method for the commercialproduction of a nicotinic alkaloid, comprising (a) providing a pluralityof cells expressing A622 and NBB1 and (b) obtaining said nicotinicalkaloid from said plurality.

In another aspect, the invention provides a method for increasingnicotine in a plant, comprising overexpressing PMT and QPT relative to acontrol plant. In one embodiment, an increased nicotine plant isproduced. In another embodiment, an increased nicotine product inproduced.

In another aspect, the invention provides a method of producing NBB1enzyme, comprising transforming a cell with an isolated nucleic moleculeencoding NBB1 and growing the transformed cell under conditions wherebyNBB1 enzyme is produced. In one embodiment, the transformed cell isselected from the group consisting of bacteria, yeast, filamentousfungi, algae, green plants, and mammalian cells.

In another aspect, the invention provides a method for increasingnicotine and yield in a Nicotiana plant, comprising a) crossing anincreased-nicotine Nicotiana plant with a high yielding Nicotiana plant;and b) selecting an increased-nicotine and yield Nicotiana progenyplant. In one embodiment, an increased nicotine and yield plant isproduced.

In one embodiment, the increased-nicotine plant is produced by: a)transforming a Nicotiana plant with a construct comprising, in the 5′ to3′ direction, a promoter operably linked to a heterologous nucleic acidencoding an enzyme that increases nicotine synthesis; b) regeneratingtransgenic Nicotiana plants from the transformed plant; and c) selectinga transgenic Nicotiana plant having increased-nicotine content relativeto a control plant. In a further embodiment, the nucleic acid isselected from the group consisting of QPT, PMT, A622, and NBB1.

In another aspect, the invention provides a method for increasingnicotine and yield in a Nicotiana plant, comprising: (a) transforming aNicotiana plant with (i) a first construct comprising, in the 5′ to 3′direction, a promoter operably linked to a heterologous nucleic acidencoding an enzyme that increases nicotine synthesis; and (ii) a secondconstruct comprising, in the 5′ to 3′ direction, a promoter operablylinked to a heterologous nucleic acid encoding an enzyme that increasesyield; (b) regenerating transgenic Nicotiana plants from the transformedplant; and (c) selecting a transgenic Nicotiana plant havingincreased-nicotine content and increased yield relative to a controlplant.

In one embodiment, the first construct comprises a nucleic acid encodingan enzyme selected from the group consisting of QPT, PMT, A622, andNBB1. In another embodiment, an increased nicotine and yield plant isproduced.

In another aspect, the invention provides a method for increasingnicotine in N. tabacum, comprising overexpressing PMT relative to acontrol plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RNA blot analysis of NBB1 expression

FIG. 2: Alignment of NBB1 with Eschscholzia californica berberine bridgeenzyme (EcBBE) (SEQ ID NOS 2 AND 33)

FIG. 3: Phylogenetic tree constructed using the sequences of NBB1polypeptide and plant BBE-like proteins

FIG. 4A: T-DNA region of pTobRD2-DEST

FIG. 4B: T-DNA region of pTobRD2-NBB1ox

FIG. 4C: T-DNA region of pTobRD2-A622ox

FIG. 4D: T-DNA region of pTobRD2-A622ox-NBB1ox

FIG. 4E: T-DNA region of pBI101H-E2113-DEST

FIG. 4F: T-DNA region of pE1235SΩ-NBB1

FIG. 5A: Immunoblot analysis of NBB1 in tobacco hairy roots

FIG. 5B: Immunoblot analysis of A622 in tobacco hairy roots

FIG. 6: Nicotine alkaloid contents in hairy roots of TobRD2-NBB1 (TN),TobRD2-A622 (TA), TobRD2-NBB1-A622 (TNA)

FIG. 7: Expression of A622 Protein in Transgenic A. belladonna

FIG. 8: Transgenic A. belladonna Expressing NBB1 and A622

FIG. 9: Nicotine synthesis in transgenic A. belladonna hairy rootsexpressing NBB1 and A622

FIG. 10: MS profile of nicotine synthesized in A. belladonna hairy rootsexpressing A622 and NBB1

FIG. 11A: T-DNA region of pA622pro-DEST

FIG. 11B: T-DNA region of pA622pro-PMTox

FIG. 11C: T-DNA region of pTobRD2-PMTox

FIG. 11D: T-DNA region of pA622pro-QPTox

FIG. 11E: T-DNA region of pTobRD2-QPTox

FIG. 11F: T-DNA region of pA622pro-PMTox-QPTox

FIG. 11G: T-DNA region of pTobRD2-PMTox-QPTox

FIG. 12: Nicotine content in leaf of tobacco plants transformed withA622pro-PMTox (AP-1 to AP-24). AG-1 to AG-4 are control plantstransformed with A622-GUS

FIG. 13: Nicotine content in leaf of tobacco plants transformed withTobRD2-PMTox (TP-1 to TP-14). TG-1 to TG-3 are control plantstransformed with TobRD2-GUS

FIG. 14: Nicotine content in leaf of tobacco plants transformed withA622pro-QPTox (AQ-1 to AQ-15). AG-1 to AG-4 are control plantstransformed with A622-GUS

FIG. 15: Nicotine content in leaf of tobacco plants transformed withTobRD2-QPTox (TQ-1 to TQ-14). TG-1 to TG-3 are control plantstransformed with TobRD2-GUS

FIG. 16: Nicotine content in leaf of tobacco plants transformed withA622pro-PMTox-QPTox (APQ-1 to APQ-27). AG-1 to AG-3 are control plantstransformed with A622-GUS

FIG. 17: Nicotine content in leaf of tobacco plants transformed withpTobRD2-PMTox-QPTox (TPQ-1 to TPQ-24). TG-1 to TG-3 are control plantstransformed with TobRD2-GUS

FIG. 18A: T-DNA region of pGWB2

FIG. 18B: T-DNA region of p35S-NBB1

FIG. 18C: T-DNA region of p35S-NBB1

FIG. 19: Immunoblot analysis of Arabidopsis thaliana lines transformedwith 35S-A622-35S-NBB1-35S-PMT cassettes.

FIG. 20: Transgenic Arabidopsis co-expressing NBB1, A622, and PMT.

FIG. 21A: Confirmation of the presence of A622 and NBB1 in recombinantbacmids

FIG. 21B: Detection of A622 and NBB1 in insect cell Sf9 cells and Ni-NTAcolumn eluates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to increasing nicotinic alkaloids inNicotiana plants, as well as to producing nicotinic alkaloids in cellsthat would not do so otherwise. As described below, the presentinventors realized that four genes, A622, NBB1, QPT and PMT, can beinfluenced to achieve an increase of nicotinic alkaloid levels inNicotiana plants. That is, overexpressing any of these four genesincreases Nicotiana nicotinic alkaloids. Further increases in Nicotiananicotinic alkaloids can be achieved by simultaneously overexpressing atleast two of the four genes, such as QPT and PMT. A622 and NBB1 can beintroduced into non-nicotine producing plants or cells, thereby toeffect their production of nicotine or related compounds.

In addition to providing methodology for increasing nicotinic alkaloidsin Nicotiana, the invention also provides for concurrently increasingnicotinic alkaloids and yield in Nicotiana. Pursuant to this aspect ofthe invention, increased nicotinic alkaloids and yield can be achievedby a combination of genetic engineering techniques and conventionalbreeding.

All technical terms employed in this specification are commonly used inbiochemistry, molecular biology and agriculture; hence, they areunderstood by those skilled in the field to which this inventionbelongs. Those technical terms can be found, for example in: MOLECULARCLONING: A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook andRussel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubel et al., GreenePublishing Associates and Wiley-Interscience, New York, 1988 (withperiodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OFMETHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 5^(th) ed., vol.1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS:A LABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1997; PRODUCTION, CHEMISTRYAND TECHNOLOGY, D. L. Davis and M. T. Nielson (eds.) Coresta, 1999(commonly referred to as “IOM Report”).

Methodology involving plant biology techniques are described here andalso are described in detail in treatises such as METHODS IN PLANTMOLEULAR BIOLOGY: A LABORATORY COURSE MANUAL, ed. Maliga et al., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995. Varioustechniques using PCR are described, for example, in Innis et al., PCRPROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, SanDiego, 1990 and in Dieffenbach and Dveksler, PCR PRIMER: A LABORATORYMANUAL, 2^(nd) ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2003. PCR-primer pairs can be derived from known sequencesby known techniques such as using computer programs intended for thatpurpose, e.g., Primer, Version 0.5, 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass. Methods for chemical synthesis ofnucleic acids are discussed, for example, in Beaucage & Caruthers,Tetra. Letts. 22: 1859-62 (1981), and Matteucci & Caruthers, J. Am.Chem. Soc. 103: 3185 (1981).

Restriction enzyme digestions, phosphorylations, ligations, andtransformations were done as described in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd ed. (1989), Cold Spring HarborLaboratory Press. All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, providesinformation to a cell from which a series of amino acids can beassembled into a specific amino acid sequence to produce an activeenzyme. Because of the degeneracy of the genetic code, certain basechanges in DNA sequence do not change the amino acid sequence of aprotein. It is therefore understood that modifications in the DNAsequences encoding A622 and NBB1, respectively, which do notsubstantially affect the functional properties of either enzyme arecontemplated.

I. Increasing Nicotinic Alkaloids in Nicotiana by Overexpressing atLeast One of A622 and NBB1

Although A622 and NBB1 were previously identified, before the presentinvention the field was wholly unaware that overexpressing at least oneof A622 or NBB1 in a Nicotiana plant increases nicotinic alkaloidcontent. Accordingly, the present invention encompasses both methodologyand constructs for increasing nicotinic alkaloid content in a Nicotianaplant, by overexpressing at least one of A622 or NBB1. Overexpressingboth A622 and NBB1 further increases nicotinic alkaloids levels in aNicotiana plant.

In the present description, an “alkaloid” is a nitrogen-containing basiccompound found in plants and produced by secondary metabolism. A“nicotinic alkaloid” is nicotine or an alkaloid that is structurallyrelated to nicotine. In the case of tobacco, nicotinic alkaloid contentand total alkaloid content are used synonymously.

Illustrative major Nicotiana nicotinic alkaloids include but are notlimited to nicotine, nornicotine, anatabine, and anabasine. Illustrativeminor Nicotiana alkaloids include but are not limited to anatalline,N-methylanatabine, N-methylanabasine, myosmine, anabaseine,N′-formylnornicotine, nicotyrine, and cotinine. Other minor nicotinicalkaloids in tobacco are reported, for example, in Hecht, S. S. et al.,Accounts of Chemical Research 12: 92-98 (1979); Tso, T. C., PRODUCTION,PHYSIOLOGY AND BIOCHEMISTRY OF TOBACCO PLANT, Ideals Inc. (Beltsville,Md.), 1990.

Many other pyridyl bases plus many derivatives of nornicotine,anatabine, and anabasine are nicotinic alkaloids that have been reportedto be present in tobacco and for purposes of the invention shall beincluded within minor Nicotiana alkaloids. Most of these so-called minornicotinic alkaloids are present in less than 50 μg/g (dry weight basis)and many others are present in nanogram amounts. Bush, L. P., et al.,“Biosynthesis and metabolism in nicotine and related alkaloids” inNICOTINE AND RELATED ALKALOIDS, J. W. Gorrod & J. Wahren (eds.) Chapman& Hall, London (1993); Bush, L. P., et al., “Alkaloid Biosynthesis” inTOBACCO PRODUCTION, CHEMISTRY AND TECHNOLOGY, D. L. Davis and M. T.Nielson (eds.) Coresta, 1999. The chemical structures of severalnicotinic alkaloids are presented, for example, in Felpin et al., J.Org. Chem. 66: 6305-312 (2001).

Nicotine is the primary alkaloid in N. tabacum, as in 50 to 60 percentof other of Nicotiana species. Depending on the variety, about 85 toabout 95 percent of total alkaloids in N. tabacum is nicotine. Bush etal. (1999), supra; Hoffmann et al., Journal of Toxicology andEnvironmental Health 41: 1-52 (1994). Based on alkaloid accumulation inthe leaves, nornicotine, anatabine, and anabasine are the other foremostalkaloids in N. tabacum. Anatabine is usually not the primary alkaloidin any Nicotiana species but does accumulate to relatively higheramounts in three species; anabasine is the primary alkaloid in fourspecies. Nornicotine is the primary alkaloid in 30 to 40 percent ofNicotiana species.

In this description, “expression” denotes the production of the proteinproduct encoded by a nucleotide sequence. “Overexpression” refers to theproduction of a protein product in a transgenic organism that exceedslevels of production in a normal or non-genetically engineered organism.As in conventional in the art, nucleotide sequences are denoted byitalicized font (e.g. PMT), whereas polypeptide sequences are notitalicized (e.g. PMT)

A622

It has been reported that A622 exhibits the same expression pattern asPMT. Shoji et al., Plant Cell Physiol. 41: 1072-76 (2000a), and PlantMol. Biol. 50: 427-40 (2002). Both A622 and PMT are expressedspecifically in roots, particularly in the cortex and endodermis of theapical parts of the roots and root hairs. Moreover, A622 and PMT have acommon pattern of expression in response to NIC regulation andmethyl-jasmonate stimulus. A622 is induced in the roots of Nicotianatabacum in response to wounding of aerial tissues. Cane et al., Func.Plant Biol. 32: 305-20 (2005). In N. glauca, A622 is induced in woundedleaves under conditions that result in QPT induction. Sinclair et al.,Func. Plant Biol. 31: 721-29 (2004).

NIC1 and NIC2 loci are two independent genetic loci in N. tabacum,formerly designated as A and B. Mutations nic1 and nic2 reduceexpression levels of nicotine biosynthesis enzymes and nicotine content,generally the nicotine content of wild type>homozygous nic2>homozygousnic1>homoyzgous nic1 and homozygous nic2 plants. Legg & Collins, Can. J.Cyto. 13: 287 (1971); Hibi et al., Plant Cell 6: 723-35 (1994); Reed &Jelesko, Plant Science 167: 1123 (2004). In this description, “nic1nic2”denotes tobacco genotypes that are homozygous for both the nic1 and thenic2 mutations.

The nucleic acid sequence of A662 (SEQ ID NO: 3) has been determined.Hibi et al. (1994), supra. The protein A622 (SEQ ID NO: 4), encoded bythis nucleic acid sequence, resembles isoflavone reductases (IFR) andcontains an NADPH-binding motif. A622 shows homology to TP7, a tobaccophenylcoumaran benzylic ether reductase (PCBER) involved in ligninbiosynthesis. Shoji et al. (2002), supra. No PCBER activity wasobserved, however, when A622 expressed in E. coli was assayed with twodifferent substrates.

Based on co-regulation of A622 and PMT and the similarity of A622 toIFR, A622 was proposed to function as a reductase in the final steps ofnicotinic alkaloid synthesis. Hibi et al. (1994); Shoji, et al. (2000a).No IFR activity was observed, however, when the protein was expressed inbacteria (id.). Heretofore there was no understanding thatoverexpressing A622 increases nicotine levels.

“A622 expression” refers to biosynthesis of a gene product encoded bySEQ ID NO: 3. “A622 overexpression” denotes an increasing of A622expression. A622 overexpression affects an increase in nicotinicalkaloid content for a plant or cell in which the overexpression occurs.A622 overexpression includes the biosynthesis of a gene product encodedby the following: full-length A622 nucleic acid sequence disclosed inHibi et al. (1994), supra (SEQ ID NO: 3), SEQ ID NO: 10, and all A622polynucleotide variants.

NBB1

The NBB1 sequence was identified as a cDNA prepared from a Nicotianasylvestris-derived cDNA library, pursuant to the protocol of Katoh etal., Proc. Japan Acad. 79 (Ser. B): 151-54 (2003). Like A622, NBB1 iscontrolled by the nicotine biosynthesis regulatory loci, NIC1 and NIC2.NBB1 and PMT have the same pattern of expression in tobacco plants. ThatNBB1 is involved in nicotine biosynthesis is indicated by the fact thatNBB1, like PMT and A622, is under the control of the NIC genes andexhibits a similar pattern of expression.

The nucleic acid sequence of NBB1 (SEQ ID NO: 1) has been determined andencodes the polypeptide sequence set forth in SEQ ID NO: 2. “NBB1expression” refers to biosynthesis of a gene product encoded by SEQ IDNO: 1. “NBB1 overexpression” denotes an increasing of NBB1 expression.NBB1 overexpression affects an increase in nicotinic alkaloid contentfor a plant or cell in which the overexpression occurs. NBB1overexpression includes biosynthesis of a gene product encoded by thefollowing: SEQ ID NO: 1, SEQ ID NO: 9, and all NBB1 polynucleotidevariants.

II. Increasing Nicotinic Alkaloids in N. tabacum by Overexpressing PMT

The only previous report demonstrating overexpression of a nicotinicbiosynthesis gene in any Nicotiana species was in N. sylvestris, wherePMT overexpression resulted in a modest 40% increase in leaf nicotine.Sato et al., Proc. Nat'l Acad. Sci. USA 98: 367-72 (2001). Whileoverexpressing a nicotinic alkaloid biosynthesis gene in one plantspecies, such as N. sylvestris, results in an increased accumulation ofsecondary metabolites, it does not necessarily follow that similaraccumulation of secondary metabolites will occur in a related species,such as N. tabacum. Saitoh et al., Phytochemistry 24: 477-80 (1985).This is especially relevant for PMT overexpression, since N. tabacumcontains five expressed PMT genes and N. sylvestris contains threeexpressed PMT genes. Hashimoto et al., Plant Mol. Biol. 37: 25-37(1998); Reichers & Timko, Plant Mol. Biol. 41: 387-401 (1999). Indeed,when the PMT gene from N. tabacum was overexpressed in Duboisia hairyroot cultures, the levels of nicotine, hyoscyamine, and scopolamine didnot increase significantly. Moyano et al., Phytochemistry 59, 697-702(2002). Likewise, overexpressing the same PMT gene in transgenic plantsand hairy root cultures of Atropa belladonna did not affect hyoscyamineand scopolamine levels. Sato et al., Proc. Nat'l Acad. Sci. USA 98:367-72 (2001); Rothe et al., J. Exp. Bot. 54: 2065-070 (2003).

In Solanaceous species, such as tobacco, it seems that the same alkaloidbiosynthesis pathway in two related plant species can be differentlyregulated and overexpression of a given gene does not necessarily leadto a similar accumulation pattern of secondary metabolites. Moyano etal., J. Exp. Bot. 54: 203-11 (2003). For example, when sixty Nicotianaspecies were analyzed, there was considerable variation in totalalkaloid content and alkaloid profile amongst the species. Saitoh etal., Phytochemistry 24: 477-80 (1985). For instance, while N. sylvestrishad the highest dry weight content of total alkaloids (the sum ofnicotine, nornicotine, anabasine and anatabine) at 29,600 μg/g or 2.96percent, N. alata contained the lowest at 20 μg/g or 0.002 percent. Theratio of nicotine to total alkaloid in the leaves of N. sylvestris wasabout 80 percent versus about 95 percent for N. tabacum L. Id. Also, theratio of nornicotine to total alkaloid in N. sylvestris leaves was 19.1percent versus 3 percent for N. tabacum L. Id. Based on these largevariations among the sixty Nicotiana species, Saitoh et al. conclude,“The amount and ratio of total and individual alkaloids present in aplant depend on the species. No clear-cut correlation between alkaloidpattern and classification of the genus Nicotiana seems to exist.” Id.at page 477.

Accordingly, the instant invention provides methodology and constructsfor increasing nicotinic alkaloids in N. tabacum by overexpressing PMT.

The nucleic acid sequence of PMT (SEQ ID NO: 7) has been determined andencodes the polypeptide sequence set forth in SEQ ID NO: 8. “PMTexpression” refers to biosynthesis of a gene product encoded by SEQ IDNO: 7. “PMT overexpression” denotes an increasing of PMT expression. PMToverexpression affects an increase in nicotinic alkaloid content for aplant or cell in which the overexpression occurs. PMT overexpressionincludes the biosynthesis of a gene product encoded by the following:full-length PMT nucleic acid sequence disclosed in Hibi et al. (1994),supra (SEQ ID NO: 7), SEQ ID NO: 12, and all PMT polynucleotidevariants.

III. Increasing Nicotinic Alkaloids in Nicotiana by Overexpressing QPTand PMT

Until now there was no knowledge that overexpressing QPT and PMTsynergistically increases nicotinic alkaloids. That is, furtherincreases in nicotine levels can be achieved by overexpressing QPT andPMT, as compared with overexpressing QPT or PMT alone. Pursuant to thisaspect of the invention, a nucleic acid construct comprising both QPTand PMT is introduced into a Nicotiana plant cell.

The nucleic acid sequence of QPT (SEQ ID NO: 5) has been determined andencodes the polypeptide sequence set forth in SEQ ID NO: 6. “QPTexpression” refers to biosynthesis of a gene product encoded by SEQ IDNO: 5. “QPT overexpression” denotes an increasing of QPT expression. QPToverexpression affects an increase in nicotinic alkaloid content for aplant or cell in which the overexpression occurs. QPT overexpressionincludes the biosynthesis of a gene product encoded by the following:full-length QPT nucleic acid sequence disclosed in U.S. Pat. No.6,423,520 (SEQ ID NO: 5), SEQ ID NO: 11, and all QPT polynucleotidevariants.

IV. Increasing Nicotinic Alkaloids in Nicotiana by Overexpressing atLeast Two or More of A622, NBB1, QPT, and PMT

While it is well-recognized that QPT plays a role in nicotinebiosynthesis, see WO 98/56923, the present invention contemplatesfurther increases in nicotine synthesis by overexpressing at least twoor more of A622, NBB1, QPT, and PMT in Nicotiana. Pursuant to thisaspect of the invention, a nucleic acid construct comprising at leasttwo of A622, NBB1, QPT, and PMT is introduced into a Nicotiana plantcell. An illustrative nucleic acid construct may comprise both QPT andA622.

V. Increasing Nicotiana Nicotinic Alkaloids and Yield

Increased nicotine plants of the invention may be produced byconventional breeding or crossing, as described by Wernsman et al., in 2PRINCIPLES OF CULTIVAR DEVELOPMENT: CROP SPECIES (Macmillan 1997). Forexample, a stable genetically engineered transformant, regenerated fromtobacco material that contains a suitable transgene, is employed tointrogress a high-nicotine trait into a desirable commerciallyacceptable genetic background, thereby obtaining a tobacco cultivar orvariety that combines a high nicotine level with said desirablebackground.

Similarly, for example, a genetically engineered plant overexpressingQPT and A622 may be produced by crossing a transgenic plantoverexpressing QPT with a transgenic plant overexpressing A622.Following successive rounds of crossing and selection, a geneticallyengineered plant having overexpressing QPT and A622 can be selected.

While any desirable gene can be introgressed into a high-nicotinevariety, there is a critical need for introducing a high nicotine traitinto a high-yielding tobacco background. Several studies indicate that“Yield improvements have been hampered by the negative relationship thatexists with nicotine concentration.” PRODUCTION, CHEMISTRY ANDTECHNOLOGY, D. L. Davis and M. T. Nielson (eds.) Coresta at page 46(1999). In his reflections of tobacco breeding, Dr. Earl Wernsmanasserts “continued selection for yield alone will soon result in apopulation whose nicotine concentration in cured leaf is so low that thetobaccos are unacceptable to industry” Wernsman, Recent Advances inTobacco Science 25: 5-35 (1999). He postulates that “genetic methods ofup-regulating nicotine synthesis may be needed to permit additionalincreases in yielding ability while maintaining nicotine concentration”Id.

Accordingly, the present invention provides a means for correcting the“negative correlation” between yield and nicotine content in Nicotianaplants by overexpressing a gene encoding a nicotine biosynthesis enzymein a high-yielding Nicotiana plant. Exemplary nicotine biosynthesisenzymes include but are not limited to QPTase, PMTase, A622, NBB1,arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADHdehydrogenase, ornithine decarboxylase (ODC), and S-adenosyl-methioninesynthetase (SAMS). Increased-nicotine plants resulting there from arethen crossed with any desirable commercially acceptable geneticbackground that maintains high yield. Suitable high-yield Nicotianaplants include but are not limited to Nicotiana tabacum cultivars K 326,NC71, NC72 and RG81. Following successive rounds of crossing andselection, a genetically engineered plant having increased nicotine andincreased yield is accordingly produced.

A further aspect of the invention provides crossing anincreased-nicotine plant with an increased-yield plant as anotherstrategy for breaking the negative correlation between nicotine contentand yield.

“Increased yield genes” encompass any gene whose expression correlateswith increased production capacity as reflected by, for example,increased photoassimilate production, increased growth rate, improvedvigor, enhanced yield, enhanced CO₂ fixation, enhanced biomass,increased seed production, improved storage, enhanced yield, increaseddisease tolerance, increased insect tolerance, increased water-stresstolerance, enhanced sweetness, improved starch composition, improvedsucrose accumulation and export, and improved response to oxidativestress compared with a wild-type control plant.

Likewise, an “increased yield plant” refers to a plant, or any portionthereof, overexpressing an “increased yield gene” and exhibits increasedproduction capacity as reflected by, for example, increasedphotoassimilate production, increased growth rate, improved vigor,enhanced yield, enhanced CO₂ fixation, enhanced biomass, increased seedproduction, improved storage, enhanced yield, increased diseasetolerance, increased insect tolerance, increased water-stress tolerance,enhanced sweetness, improved starch composition, improved sucroseaccumulation and export, and improved response to oxidative stresscompared with a wild-type control plant.

For example, and in no way limiting the invention, an increased yieldplant can be produced by overexpressing a pathogenesis-related (PR)gene. It has been shown that overexpressing a maize PRms gene, intobacco produced transgenic tobacco plants having enhanced biomass andseed production. Murillo et al., Plant J. 36: 330-41 (2003). Likewise,an increased yield plant can be produced by overexpressing a geneencoding a Calvin cycle enzyme. Tamoi et al. Plant Cell Physiol.47(3)380-390 (2006). Tobacco plants overexpressing, for example, acyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase displayedenhanced photosynthetic efficiency and growth efficiency compared withwild-type tobacco. Miyagawa et al., Nature Biotech. 19: 965-69 (2001).

The present invention also contemplates producing a plant havingincreased yield and increased nicotine by overexpressing a gene encodinga nicotine biosynthesis enzyme, such as QPT, PMT, A622, or NBB1, andoverexpressing an increased yield gene, such as PRms,fructose-1,6-/sedoheptulose-1,7-bisphosphatase,fructose-1,6-bisphosphatase, and sedoheptulose-1,7-bisphosphatase,sedoheptulose-1,7-bisphosphatase in the same plant or cell.

VI. Producing Nicotinic Alkaloids and Related Compounds in Non-NicotineProducing Cells

A622 and NBB1 can be introduced into a non-nicotine producing plant orcell, thereby producing nicotine or related compounds in an organism orcell that does not produce these compounds otherwise. A variety ofproducts can be produced from these engineered organisms and cells,including nicotine, nicotine analogs, and nicotine biosynthesis enzymes.

A “non-nicotine producing plant” refers to any plant that does notproduce nicotine or related nicotinic alkaloids. Illustrativenon-nicotine producing plants include but are not limited to Atropabelladonna and Arabidopsis thaliana.

“Non-nicotine producing cells” refers to cells from any organism thatdoes not produce nicotine or related nicotinic alkaloids. Illustrativecells include but are not limited to plant cells, such as Atropabelladonna, Arabidopsis thaliana, as well as insect, mammalian, yeast,fungal, algal, or bacterial cells.

A “nicotine analog” has the basic structure of nicotine but may, forexample, have different ring substituents. For example, a nicotineanalog may substitute a hydrogen (—H) for the methyl group (—CH₃)thereby producing nornicotine, which is an analog of nicotine. Inaddition to sharing a similar structure with nicotine, nicotine analogsmay provide similar physiological effects. Cotinine, for example, hasbeen cited for its positive effects on improving concentration andmemory and, accordingly, is a nicotine analog. Accordingly, nicotineanalogs are defined broadly to cover any and all compounds havingsimilar structural and functional activity to nicotine.

VII. Synthesis of Compounds Using Novel Enzymes

Recently, there has been great interest in synthesizing nicotine analogsthat target nicotine receptors and provide therapeutic effects forneurogenerative diseases and cognitive disabilities. For example,Targacept, a pharmaceutical company formed as a spinout from R.J.Reynolds Tobacco Company, endeavors to develop and commercializenicotine analog drugs based on selective activation of neuronalnicotinic acetylcholine receptors (NNRs). Because the present inventionprovides a novel nicotine biosynthesis enzyme, there may be value inusing NBB1 alone, or NBB1 and A622, for developing novel nicotineanalogs. For example, using the inventive methods and constructs, anicotinic alkaloid analog can be produced by providing a nicotine analogprecursor in a cell culture system.

Additionally, the inventive enzymes may be used for in vitro synthesisof nicotine and related compounds. That is, recombinant A622 and NBB1can be used for the synthesis or partial synthesis of a nicotinicalkaloid and a nicotinic alkaloid analog.

Nicotinic Alkaloid Biosynthesis Sequences

Nicotinic alkaloid biosynthesis genes have been identified in severalplant species, exemplified by Nicotiana plants. Accordingly, the presentinvention embraces any nucleic acid, gene, polynucleotide, DNA, RNA,mRNA, or cDNA molecule that is isolated from the genome of a plantspecies, or produced synthetically, that increases Nicotiana nicotinicalkaloid biosynthesis. Additionally, expression of such nicotinicalkaloid biosynthesis sequence produces nicotinic alkaloids in anon-nicotine producing cell, such as an insect cell. The DNA or RNA maybe double-stranded or single-stranded. Single-stranded DNA may be thecoding strand, also known as the sense strand, or it may be thenon-coding strand, also called the anti-sense strand.

It is understood that NBB1, A622, QPT, and PMT include the sequences setforth in SEQ ID NO: 1, 9, 3, 10, 5, 11, 7, and 12, respectively, as wellas nucleic acid molecules comprised of variants of SEQ ID NO: 1, 9, 3,10, 5, 11, 7, and 12, with one or more bases deleted, substituted,inserted, or added, which variant codes for a polypeptide with nicotinicalkaloid biosynthesis activity. Accordingly, sequences having “basesequences with one or more bases deleted, substituted, inserted, oradded” retain physiological activity even when the encoded amino acidsequence has one or more amino acids substituted, deleted, inserted, oradded. Additionally, multiple forms of A622, NBB1, QPTase, and PMTasemay exist, which may be due to post-translational modification of a geneproduct, or to multiple forms of the respective PMT, QPT, A622, or NBB1genes. Nucleotide sequences that have such modifications and that codefor a nicotinic alkaloid biosynthesis enzyme are included within thescope of the present invention.

For example, the poly A tail or 5′- or 3′-end, nontranslation regionsmay be deleted, and bases may be deleted to the extent that amino acidsare deleted. Bases may also be substituted, as long as no frame shiftresults. Bases also may be “added” to the extent that amino acids areadded. It is essential, however, that any such modification does notresult in the loss of nicotinic alkaloid biosynthesis enzyme activity. Amodified DNA in this context can be obtained by modifying the DNA basesequences of the invention so that amino acids at specific sites aresubstituted, deleted, inserted, or added by site-specific mutagenesis,for example. Zoller & Smith, Nucleic Acid Res. 10: 6487-500 (1982).

A nicotinic alkaloid biosynthesis sequence can be synthesized ab initiofrom the appropriate bases, for example, by using an appropriate proteinsequence disclosed herein as a guide to create a DNA molecule that,though different from the native DNA sequence, results in the productionof a protein with the same or similar amino acid sequence. This type ofsynthetic DNA molecule is useful when introducing a DNA sequence into anon-plant cell, coding for a heterologous protein, that reflectsdifferent (non-plant) codon usage frequencies and, if used unmodified,can result in inefficient translation by the host cell.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in a DNAconstruct are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells or DNAmolecules that are purified, partially or substantially, in solution.Isolated RNA molecules include in vitro RNA transcripts of the DNAmolecules of the present invention. Isolated nucleic acid molecules,according to the present invention, further include such moleculesproduced synthetically.

“Exogenous nucleic acid” refers to a nucleic acid, DNA or RNA, which hasbeen introduced into a cell (or the cell's ancestor) through the effortsof humans. Such exogenous nucleic acid may be a copy of a sequence whichis naturally found in the cell into which it was introduced, orfragments thereof.

In contrast, “endogenous nucleic acid” refers to a nucleic acid, gene,polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is present in thegenome of a plant or organism that is to be genetically engineered. Anendogenous sequence is “native” to, i.e., indigenous to, the plant ororganism that is to be genetically engineered.

“Heterologous nucleic acid” refers to a nucleic acid, DNA or RNA, whichhas been introduced into a cell (or the cell's ancestor) which is not acopy of a sequence naturally found in the cell into which it isintroduced. Such heterologous nucleic acid may comprise segments thatare a copy of a sequence which is naturally found in the cell into whichit has been introduced, or fragments thereof.

A “chimeric nucleic acid” comprises a coding sequence or fragmentthereof linked to a transcription initiation region that is differentfrom the transcription initiation region with which it is associated incells in which the coding sequence occurs naturally.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer, such as the Model 373 from Applied Biosystems, Inc.Therefore, as is known in the art for any DNA sequence determined bythis automated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 95% identical, more typically at least about96% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence may be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

For the purpose of the invention, two sequences hybridize when they forma double-stranded complex in a hybridization solution of 6× SSC, 0.5%SDS, 5× Denhardt's solution and 100 μg of non-specific carrier DNA. SeeAusubel et al., supra, at section 2.9, supplement 27 (1994). Sequencesmay hybridize at “moderate stringency,” which is defined as atemperature of 60° C. in a hybridization solution of 6× SSC, 0.5% SDS,5× Denhardt's solution and 100 μg of non-specific carrier DNA. For “highstringency” hybridization, the temperature is increased to 68° C.Following the moderate stringency hybridization reaction, thenucleotides are washed in a solution of 2× SSC plus 0.05% SDS for fivetimes at room temperature, with subsequent washes with 0.1× SSC plus0.1% SDS at 60° C. for 1 h. For high stringency, the wash temperature isincreased to 68° C. For the purpose of the invention, hybridizednucleotides are those that are detected using 1 ng of a radiolabeledprobe having a specific radioactivity of 10,000 cpm/ng, where thehybridized nucleotides are clearly visible following exposure to X-rayfilm at −70° C. for no more than 72 hours.

The present application is directed to such nucleic acid molecules whichare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a nucleic acid sequence described in any of SEQ IDNO: 1, 9, 3, 10, 5, 11, 7, and 12. Preferred are nucleic acid moleculeswhich are at least 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid sequence shown in any of SEQ ID NO: 1, 9, 3, 10, 5, 11, 7,and 12. Differences between two nucleic acid sequences may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotidesequence refers to a comparison made between two molecules usingstandard algorithms well known in the art and can be determinedconventionally using publicly available computer programs such as theBLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-402(1997).

The present invention further provides nucleic acid molecules comprisingthe nucleotide sequence of SEQ ID NOs.: 1, 1B, 3, 3B, 5, 5B, 7, and 7B,respectively, which encode an active nicotine biosynthesis enzyme,wherein the enzyme has amino acid sequence that corresponds to SEQ IDNO.: 2, 4, 6, and 8, respectively, and wherein the protein of theinvention encompasses amino acid substitutions, additions and deletionsthat do not alter the function of the nicotine biosynthesis enzyme.

A “variant” is a nucleotide or amino acid sequence that deviates fromthe standard, or given, nucleotide or amino acid sequence of aparticular gene or protein. The terms “isoform,” “isotype,” and “analog”also refer to “variant” forms of a nucleotide or an amino acid sequence.An amino acid sequence that is altered by the addition, removal, orsubstitution of one or more amino acids, or a change in nucleotidesequence, may be considered a “variant” sequence. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. A variant may have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues may be substituted, inserted,or deleted may be found using computer programs well known in the artsuch as Vector NTI Suite (InforMax, MD) software. “Variant” may alsorefer to a “shuffled gene” such as those described in Maxygen-assignedpatents.

Nucleic Acid Constructs

In accordance with one aspect of the invention, a sequence thatincreases nicotinic alkaloid biosynthesis is incorporated into a nucleicacid construct that is suitable for plant or cell transformation. Thus,such a nucleic acid construct can be used to overexpress at least one ofA622, NBB1, PMT, and QPT in a plant, as well as express A622 and NBB1,for example, in a non-nicotine producing cell.

Recombinant nucleic acid constructs may be made using standardtechniques. For example, the DNA sequence for transcription may beobtained by treating a vector containing said sequence with restrictionenzymes to cut out the appropriate segment. The DNA sequence fortranscription may also be generated by annealing and ligating syntheticoligonucleotides or by using synthetic oligonucleotides in a polymerasechain reaction (PCR) to give suitable restriction sites at each end. TheDNA sequence then is cloned into a vector containing suitable regulatoryelements, such as upstream promoter and downstream terminator sequences.

An important aspect of the present invention is the use of nucleic acidconstructs wherein a nicotinic alkaloid biosynthesis-encoding sequenceis operably linked to one or more regulatory sequences, which driveexpression of the nicotinic alkaloid biosynthesis-encoding sequence incertain cell types, organs, or tissues without unduly affecting normaldevelopment or physiology.

“Promoter” connotes a region of DNA upstream from the start oftranscription that is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “constitutivepromoter” is one that is active throughout the life of the plant andunder most environmental conditions. Tissue-specific, tissue-preferred,cell type-specific, and inducible promoters constitute the class of“non-constitutive promoters.” “Operably linked” refers to a functionallinkage between a promoter and a second sequence, where the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. In general, “operably linked”means that the nucleic acid sequences being linked are contiguous.

Promoters useful for expression of a nucleic acid sequence introducedinto a cell to increase expression of A622, NBB1, PMTase, or QPTase maybe constitutive promoters, such as the cauliflower mosaic virus (CaMV)35S promoter, or tissue-specific, tissue-preferred, cell type-specific,and inducible promoters. Preferred promoters include promoters which areactive in root tissues, such as the tobacco RB7promoter (Hsu et al.Pestic. Sci. 44: 9-19 (1995); U.S. Pat. No. 5,459,252), maize promoterCRWAQ81 (US published patent application 20050097633); the ArabidopsisARSK1 promoter (Hwang and Goodman, Plant J 8:37-43 (1995)), the maizeMR7 promoter (U.S. Pat. No. 5,837,848), the maize ZRP2 promoter (U.S.Pat. No. 5,633,363), the maize MTL promoter (U.S. Pat. Nos. 5,466,785and 6,018,099) the maize MRS1, MRS2, MRS3, and MRS4 promoters (U.S. Pat.App. 20050010974), an Arabidopsis cryptic promoter (U.S. Pat. App.20030106105) and promoters that are activated under conditions thatresult in elevated expression of enzymes involved in nicotinebiosynthesis such as the tobacco RD2 promoter (U.S. Pat. No. 5,837,876),PMT promoters (Shoji T. et al., Plant Cell Physiol. 41: 831-39 (2000b);WO 2002/038588) or an A622 promoter (Shoji T. et al., Plant Mol Biol.50: 427-40 (2002)).

The vectors of the invention may also contain termination sequences,which are positioned downstream of the nucleic acid molecules of theinvention, such that transcription of mRNA is terminated, and polyAsequences added. Exemplary of such terminators are the cauliflowermosaic virus (CaMV) 35S terminator and the nopaline synthase gene (Tnos)terminator. The expression vector also may contain enhancers, startcodons, splicing signal sequences, and targeting sequences.

Expression vectors of the invention may also contain a selection markerby which transformed cells can be identified in culture. The marker maybe associated with the heterologous nucleic acid molecule, i.e., thegene operably linked to a promoter. As used herein, the term “marker”refers to a gene encoding a trait or a phenotype that permits theselection of, or the screening for, a plant or cell containing themarker. In plants, for example, the marker gene will encode antibioticor herbicide resistance. This allows for selection of transformed cellsfrom among cells that are not transformed or transfected.

Examples of suitable selectable markers include adenosine deaminase,dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidinekinase, xanthine-guanine phospho-ribosyltransferase, glyphosate andglufosinate resistance, and amino-glycoside 3′-O-phosphotransferase(kanamycin, neomycin and G418 resistance). These markers may includeresistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.The construct may also contain the selectable marker gene Bar thatconfers resistance to herbicidal phosphinothricin analogs like ammoniumgluphosinate. Thompson et al., EMBO J. 9: 2519-23 (1987). Other suitableselection markers are known as well.

Visible markers such as green florescent protein (GFP) may be used.Methods for identifying or selecting transformed plants based on thecontrol of cell division have also been described. See WO 2000/052168and WO 2001/059086.

Replication sequences, of bacterial or viral origin, may also beincluded to allow the vector to be cloned in a bacterial or phage host.Preferably, a broad host range prokaryotic origin of replication isused. A selectable marker for bacteria may be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other nucleic acid sequences encoding additional functions may also bepresent in the vector, as is known in the art. For instance, whenAgrobacterium is the host, T-DNA sequences may be included to facilitatethe subsequent transfer to and incorporation into plant chromosomes.

Plants for Genetic Engineering

The present invention comprehends the genetic manipulation of aNicotiana plant for increasing nicotinic alkaloid synthesis viaintroducing a polynucleotide sequence that encodes an enzyme in thepathway for nicotinic alkaloid synthesis. Additionally, the inventionprovides methods for producing nicotinic alkaloids and related compoundsin non-nicotine producing plants, such as Arabidopsis thaliana andAtropa belladonna.

“Genetically engineered” (GE) encompasses any methodology forintroducing a nucleic acid or specific mutation into a host organism.For example, a tobacco plant is genetically engineered when it istransformed with a polynucleotide sequence that increases expression ofa gene, such as A622 or NBB1, and thereby increases nicotine levels. Incontrast, a tobacco plant that is not transformed with a polynucleotidesequence is a control plant and is referred to as a “non-transformed”plant.

In the present context, the “genetically engineered” category includes“transgenic” plants and cells (see definition, infra), as well as plantsand cells produced by means of targeted mutagenesis effected, forexample, through the use of chimeric RNA/DNA oligonucleotides, asdescribed by Beetham et al., Proc. Nat'l. Acad. Sci. USA 96: 8774-8778(1999) and Zhu et al., loc. cit. at 8768-8773, or so-called“recombinagenic olionucleobases,” as described in PCT application WO03/013226. Likewise, a genetically engineered plant or cell may beproduced by the introduction of a modified virus, which, in turn, causesa genetic modification in the host, with results similar to thoseproduced in a transgenic plant, as described herein. See, e.g., U.S.Pat. No. 4,407,956. Additionally, a genetically engineered plant or cellmay be the product of any native approach (i.e., involving no foreignnucleotide sequences), implemented by introducing only nucleic acidsequences derived from the host species or from a sexually compatiblespecies. See, e.g., U.S. published application No. 2004/0107455.

“Plant” is a term that encompasses whole plants, plant organs (e. g.leaves, stems, roots, etc.), seeds, differentiated or undifferentiatedplant cells, and progeny of the same. Plant material includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissues, leaves, roots, shoots, stems, fruit, gametophytes,sporophytes, pollen, and microspores. The class of plants which can beused in the present invention is generally as broad as the class ofhigher plants amenable to genetic engineering techniques, including bothmonocotyledonous and dicotyledonous plants, as well as gymnosperms. Apreferred nicotine-producing plant includes Nicotiana, Duboisia,Anthocericis and Salpiglessis genera in the Solanaceae or the Ecliptaand Zinnia genera in the Compositae.

“Tobacco” refers to any plant in the Nicotiana genus that producesnicotinic alkaloids. Tobacco also refers to products comprising materialproduced by a Nicotiana plant, and therefore includes, for example,expanded tobacco, reconstituted tobacco, cigarettes, cigars, chewingtobacco or forms of smokeless tobacco, snuff and snus made from GEincreased-nicotine tobacco. Examples of Nicotiana species include butare not limited to the following: Nicotiana acaulis, Nicotianaacuminata, Nicotiana acuminata var. multiflora, Nicotiana africana,Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotianaattenuata, Nicotiana benavidesii, Nicotiana benthamiana, Nicotianabigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotianaclevelandii, Nicotiana cordifolia, Nicotiana corymbosa, Nicotianadebneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans,Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotianagossei, Nicotiana hybrid, Nicotiana ingulba, Nicotiana kawakamii,Nicotiana knightiana, Nicotiana langsdorffii, Nicotiana linearis,Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon,Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotianaobtusifolia, Nicotiana occidentalis, Nicotiana occidentalis subsp.hesperis, Nicotiana otophora, Nicotiana paniculata, Nicotianapauciflora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotianaquadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotianarosulata, Nicotiana rosulata subsp. ingulba, Nicotiana rotundifolia,Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotianasolanifolia, Nicotiana spegazzinii, Nicotiana stocktonii, Nicotianasuaveolens, Nicotiana sylvestris, N. tabacum, Nicotiana thyrsiflora,Nicotiana tomentosa, Nicotiana tomentosiformis, Nicotiana trigonophylla,Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotianawigandioides, and Nicotiana×sanderae.

In the present description, “tobacco hairy roots” refers to tobaccoroots that have T-DNA from an Ri plasmid of Agrobacterium rhizogenesintegrated in the genome and grow in culture without supplementation ofauxin and other phytohormones. Tobacco hairy roots produce nicotinicalkaloids as roots of a tobacco plant do. These types of roots arecharacterized by fast growth, frequent branching, plagiotropism, and theability to synthesize the same compounds as the roots of the intactplant. David et al., Biotechnology 2: 73-76. (1984). Roots of Solanaceaeplants are the main site of tropane alkaloid biosynthesis, and hencehairy root cultures also are capable of accumulating high levels ofthese metabolites. For example, see Oksman-Caldentey & Arroo,“Regulation of tropane alkaloid metabolism in plants and plant cellcultures,” in METABOLIC ENGINEERING OF PLANT SECONDARY METABOLISM 253-81(Kluwer Academic Publishers, 2000).

Non-Nicotine Producing Cells for Genetic Engineering

The invention contemplates genetically engineering “non-nicotineproducing cells” with a nucleic acid sequence encoding an enzymeinvolved in the production of nicotinic alkaloids. Non-nicotineproducing cells refer to a cell from any organism that does not producenicotine. Illustrative cells include but are not limited to plant cells,such as Atropa belladonna, Arabidopsis thaliana, as well as insect,mammalian, yeast, fungal, algal, or bacterial cells. Suitable host cellsare discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

“Insect cell” refers to any insect cell that can be transformed with agene encoding a nicotine biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative insect cells include Sf9 cells (ATCC CRL 1711).

“Fungal cell” refers to any fungal cell that can be transformed with agene encoding a nicotine biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative fungal cells include yeast cells such as Saccharomycescerivisae (Baldari, et al., 1987. EMBO J. 6: 229-234) and Pichiapastoris (e.g. P. pastoris KM714 available from Invitrogen). Cells offilamentous fungi such as Aspergillus and Trichoderma may also be used.Archer, et al., Antonie van Leeuwenhoek 65: 245-250 (2004).

“Bacterial cell” refers to any bacterial cell that can be transformedwith a gene encoding a nicotinic alkaloid biosynthesis enzyme and iscapable of expressing in recoverable amounts the enzyme or its products.Illustrative bacterial cells include E. coli, such as E. coli strainM15/rep4, which is available commercially from QIAGEN.

“Mammalian cell” refers to any mammalian cell that can be transformedwith a gene encoding a nicotine biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative mammalian cells include Chinese hamster ovary cells (CHO)or COS cells. Mammalian cells may also include a fertilized oocyte or anembryonic stem cell into which nicotinic alkaloid biosynthesisenzyme-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals. Examples of systems forregulated expression of proteins in mamamlian cells include Clontech'sTet-Off and Tet-On gene expression systems and similar systems. Gossenand Bujard, Proc. Natl. Acad. Sci. USA 89: 55475551 (1992).

“Algae cell” refers to any algae species that can be transformed with agene encoding a nicotine biosynthesis enzyme without adversely affectingnormal algae development or physiology. Illustrative algae cells includeChlamydomonas reinhardtii (Mayfield and Franklin, Vaccine 23: 1828-1832(2005).

Because production of nicotinic alkaloids in an insect cell couldadversely affect insect growth and development, an inducible expressionsystem may mitigate adverse affects. For example, insect cells may befirst grown under non-inducing conditions to a desired state and thenexpression of the enzyme is induced.

Additionally, cells expressing nicotinic alkaloid biosynthesis genes maybe supplied with precursors to increase substrate availability fornicotinic alkaloid synthesis. Cells may be supplied with analogs ofprecursors which may be incorporated into analogs of naturally occurringnicotinic alkaloids.

Transformation and Selection

While nicotine is the major alkaloid in N. tabacum and some otherspecies in the Nicotiana genus, other plants have nicotine-producingability, including, for example, Duboisia, Anthocericis and Salpiglessisgenera in the Solanaceae, and Eclipta and Zinnia genera in theCompositae. Using the inventive constructs and methods, nicotine may beproduced in non-nicotine producing plants, such as Atropa belladonna andArabidopsis thaliana, and cells, such as insect, fungal, and bacterialcells.

For the purposes of this description, a plant or non-nicotine producingcell, such as a fungal cell, may be transformed with a plasmidcomprising one or more sequences, each operably linked to a promoter.For example, an illustrative vector may comprise a QPT sequence operablylinked to a promoter. Likewise, the plasmid may comprise a QPT sequenceoperably linked to a promoter and an A622 sequence operably linked to apromoter. Alternatively, a plant or non-nicotine producing cell may betransformed with more than one plasmid. For example, a plant ornon-nicotine producing cell may be transformed with a first plasmidcomprising a QPT sequence operably linked to a promoter, which isdistinct from a second plasmid comprising an A622 or NBB1 sequence. Ofcourse, the first and second plasmids or portions thereof are introducedinto the same cell.

Plant Transformation

“Transgenic plant” refers to a plant that comprises a nucleic acidsequence that also is present per se in another organism or species orthat is optimized, relative to host codon usage, from another organismor species. Both monocotyledonous and dicotyledonous angiosperm orgymnosperm plant cells may be transformed in various ways known to theart. For example, see Klein et al., Biotechnology 4: 583-590 (1993);Bechtold et al., C. R. Acad. Sci. Paris 316:1194-1199 (1993); Bent etal., Mol. Gen. Genet. 204:383-396 (1986); Paszowski et al., EMBO J. 3:2717-2722 (1984); Sagi et al., Plant Cell Rep. 13: 262-266 (1994).Agrobacterium species such as A. tumefaciens and A. rhizogenes can beused, for example, in accordance with Nagel et al., Microbiol Lett 67:325 (1990). Additionally, plants may be transformed by Rhizobium,Sinorhizobium or Mesorhizobium transformation. Broothaerts et al.,Nature 433:629-633 (2005).

For example, Agrobacterium may be transformed with a plant expressionvector via, e.g., electroporation, after which the Agrobacterium isintroduced to plant cells via, e.g., the well known leaf-disk method.Additional methods for accomplishing this include, but are not limitedto, electroporation, particle gun bombardment, calcium phosphateprecipitation, and polyethylene glycol fusion, transfer into germinatingpollen grains, direct transformation (Lorz et al., Mol. Genet. 199:179-182 (1985)), and other methods known to the art. If a selectionmarker, such as kanamycin resistance, is employed, it makes it easier todetermine which cells have been successfully transformed. Marker genesmay be included within pairs of recombination sites recognized byspecific recombinases such as cre or flp to facilitate removal of themarker after selection. See U. S. published application No.2004/0143874.

Transgenic plants without marker genes may be produced using a secondplasmid comprising a nucleic acid encoding the marker, distinct from afirst plasmid that comprises an A622 or NBB1 sequence. The first andsecond plasmids or portions thereof are introduced into the same plantcell, such that the selectable marker gene that is transientlyexpressed, transformed plant cells are identified, and transformedplants are obtained in which the A622 or NBB1 sequence is stablyintegrated into the genome and the selectable marker gene is not stablyintegrated. See U. S. published application No. 2003/0221213. The firstplasmid that comprises an A622 or NBB1 sequence may optionally be abinary vector with a T-DNA region that is completely made up of nucleicacid sequences present in wild-type non-transgenic N. tabacum orsexually compatible Nicotiana species.

The Agrobacterium transformation methods discussed above are known to beuseful for transforming dicots. Additionally, de la Pena et al., Nature325: 274-276 (1987), Rhodes et al., Science 240: 204-207 (1988), andShimamato et al., Nature 328: 274-276 (1989) have transformed cerealmonocots using Agrobacterium. Also see Bechtold et al., C.R. Acad. Sci.Paris 316 (1994), illustrating vacuum infiltration forAgrobacterium-mediated transformation.

Methods of regenerating a transgenic plant from a transformed cell orculture vary according to the plant species but are based on knownmethodology. For example, methods for regenerating of transgenic tobaccoplants are well-known. Genetically engineered plants are selected thathave increased expression of at least one of A622, NBB1, PMT, and QPT.Additionally, the inventive genetically engineered plants may haveincreased nicotine levels and yield.

Non-Nicotine Producing Cell Transformation

Constructs according to the invention may be used to transform any cell,using a suitable transformation technique, such asAgrobacterium-mediated transformation for plant cells, particlebombardment, electroporation, and polyethylene glycol fusion, calciumphosphate transfection, DEAE-dextran mediated transfection, or cationiclipid-mediated transfection.

Non-nicotine producing cells may be transformed with nucleic acidconstructs of the present invention without the use of a selectable orvisible marker and transgenic organisms may be identified by detectingthe presence of the introduced construct. The presence of a protein,polypeptide, or nucleic acid molecule in a particular cell can bemeasured to determine if, for example, a cell has been successfullytransformed or transfected. For example, and as routine in the art, thepresence of the introduced construct can be detected by PCR or othersuitable methods for detecting a specific nucleic acid or polypeptidesequence. Additionally, transformed cells may be identified byrecognizing differences in the growth rate or a morphological feature ofa transformed cell compared to the growth rate or a morphologicalfeature of a non-transformed cell that is cultured under similarconditions. See WO 2004/076625.

For the purposes of the present description, genetically engineeredcells are selected that express A622 and NBB1 heterologously.

Quantifying Nicotinic Alkaloid Content

Genetically engineered plants and cells are characterized by increasednicotinic alkaloid content. Similarly, transformed non-nicotineproducing cells are characterized by nicotinic alkaloid production.

In describing a plant of the invention, the phrase “increased nicotineor nicotinic alkaloid content” refers to an increase in the amount ofnicotinic alkaloid in the plant or cell when compared with anon-transformed control plant or cell. “Increased nicotine plant”encompasses a genetically engineered plant that has an increase innicotine content greater than 10%, and preferably greater than 50%,100%, or 200% of the nicotine content of a control plant of the samespecies or variety.

A successfully transformed non-nicotine producing cell is characterizedby nicotinic alkaloid synthesis. For example, a transformed non-nicotineproducing cell may produce nicotine, whereas a non-transformed controlcell does not.

A quantitative increase in nicotinic alkaloid levels can be assayed byseveral methods, as for example by quantification based on gas-liquidchromatography, high performance liquid chromatography,radio-immunoassays, and enzyme-linked immunosorbent assays. In thepresent invention, nicotinic alkaloid levels were measured by gas-liquidchromatography equipped with a capillary column and an FID detector, asdescribed in Hibi et al., Plant Physiology 100: 826-35 (1992).

Quantifying Yield

Genetically engineered plants and cells of the invention arecharacterized by increased nicotinic alkaloid content and yield.Nicotinic alkaloid production in the genetically engineered plants ispreferably achieved by expressing a nicotine biosynthesis pathway gene,such as A622, NBB1, PMT, or QPT.

In describing a plant of the invention, the phrase “increased yield” or“high yielding” refers to an increase in the amount of yield of a plantor crop of said plant when compared to an increased-nicotine controlplant or crop of said plant. “Increased yield plant” encompasses agenetically engineered plant that yields the same as a plant or crop orsaid plant as an increased-nicotine plant or crop of said plant,preferably greater than 110%, and more preferably greater than 125% ofthe yield of a nicotine-enriched control plant of the same species orvariety.

A quantitative increase in photosynthetic efficiency can be assayed byseveral methods, as for example by quantifying photosynthetic rates,such as gas exchange and CO₂ fixation, and chlorophyll florescence.Miyagawa et al., Plant Cell Physiol. 41, 311-320 (2000). Photosyntheticrates may also be quantified by measuring metabolite and carbohydratelevels as described by Leegood, Carbon Metabolism In Photosynthesis andproduction in a changing environment: a field and laboratory manual (edsHall, Scurlock, Bolhar-Nordenkampf, Leegood, & Long) 247-267 (Chapman &Hall, London; 1993). Alternatively, photosynthetic activity may becalculated based on enzyme activity, such as Rubisco activity. Portis,A. R. J. Exp. Bot. 46:1285-1291 (1995).

Of course, increased yield can be determined by measuring more readilydiscernible characteristics, including but not limited to plant height,weight, leaf size, time to flowering, number of seeds produced, and seedweight.

Increased-Nicotine Products

The present invention provides a genetically engineered plant havingincreased-nicotine levels, as well as a genetically engineerednon-nicotine producing cell that produces nicotine or related compounds,where said cell is derived from an organism that does not producenicotine. A variety of products may be made from such a geneticallyengineered plant. Likewise, products can be made from cells that aregenetically engineered for production of nicotine or related compounds.

Herbivore-Resistant Plant

Nicotine serves as a natural pesticide which helps protect tobaccoplants from damage by pests. It has been show that conventionally bredor transgenic low-nicotine tobacco have increased susceptibility toinsect damage. Legg, P. D., et al., Can. J. Cyto., 13:287-291 (1971);Voelckel, C., et al., Chemoecology 11:121-126 (2001); Steppuhn, A., etal., PLoS Biol, 2(8): e217: 1074-1080 (2004). Using the inventivemethods and constructs, increased-nicotine plants may be produced thathave increased resistance to insect and other pest damage. Similarly,increased pest resistance may achieved in non-nicotine producing plants,such as A. belladonna and A. thaliana, that are genetically engineeredaccording to the present invention to produce nicotine.

Increased-Nicotine Tobacco Products

The inventive constructs and methods may be used to produce, forexample, cigarettes, cigars, and other traditional tobacco products suchas snuff and snus. Additionally, increased-nicotine cigarettes may beproduced that have reduced-exposure to smoke components, such as tar,yet have similar or increased nicotine deliveries as conventionalcigarettes.

In the present description, an increased-nicotine tobacco product may bein the form of leaf tobacco, shredded tobacco, cut rag tobacco, groundtobacco, reconstituted tobacco, expanded or puffed tobacco and tobaccofractions including, for example, nicotine. An increased-nicotinetobacco product may include cigarettes, cigars, pipe tobaccos, and anyform of smokeless tobacco such as snuff, snus, or chewing tobacco.

Blending different tobacco types or cultivars within a tobacco productsuch as a cigarette is common in tobacco art. It will therefore beappreciated that increased-nicotine tobacco could be blended at anypercentage with non-transformed tobacco to obtain any level of desirednicotine content, up to the nicotine content of the increased nicotinetobacco utilized, to manufacture a tobacco product.

Increased nicotine cigarettes are particularly advantageous becausestudies demonstrate that when nicotine is increased, smokers inhale lesstar and carbon monoxide. See Armitage et al., Psychopharmacology96:447-453 (1988); Fagerström, Psychopharmacology 77:164-167 (1982);Russell, Nicotine and Public Health 15:265-284 (2000) and Woodman etal., European Journal of Respiratory Disease 70:316-321 (1987).

Cigarette smoke is an extremely complex mixture of more than 4,000different compounds. Green & Rodgman, Recent Advances in Tobacco Science22: 131-304 (1996); IOM Report, page 9 of executive summary. Cigarettesmoke is made up of two phases: a particulate phase, which is commonlycalled “tar” or total particulate matter, and a vapor phase, whichcontains gases and semi-volatile compounds. A common definition for“tar” is “nicotine-free dry smoke” or “nicotine-free dry particulatematter” (NFDPM) captured by a Cambridge pad when a cigarette is machinesmoked. More specifically, “tar” is the total particulate matterisolated from smoke, excluding water and nicotine. Tar makes up lessthan ten percent of the weight of cigarette smoke. Yet, it is the tarcomponent that contains the majority of the most harmful smokecompounds.

Analytical methods combined with sensitive biological assays have led tothe identification of 69 carcinogens in tobacco smoke. See THE CHANGINGCIGARETTE: CHEMICAL STUDIES AND BIOASSAYS, Chapter 5, Smoking andTobacco Control Monograph No. 13 (NIH Pub. No. 02-5074, October 2001).It has become clear to researchers, however, that not all components ofcigarette smoke have equal toxicity. Notably, the first U.S. SurgeonGeneral's report on smoking in 1964 came to the conclusion that nicotinewas probably not toxic at the levels inhaled by smokers, with theimplication that the source of the primary pharmacologic reward tosmokers was not of immediate concern. The Surgeon General's 1964 reportstated, at page 74, that “[t]here is no acceptable evidence thatprolonged exposure to nicotine creates either dangerous functionalchanges of an objective nature or degenerative diseases.”

In fact, the U.S. Food and Drug Administration allows the sale ofnicotine replacement products such as patches and chewing gum for use insmoking cessation therapy. These products may deliver more nicotine inone day than a pack of cigarettes. Page 167 of the IOM Report states,“Many studies of nicotine suggest that nicotine is unlikely to be acancer-causing agent in humans or, at worst, that its carcinogenicitywould be trivial compared to that of other components of tobacco. Theconsideration of nicotine as a carcinogenic agent, if at all, is trivialcompared to the risk of other tobacco constituents.”

Cigarettes are generally rated by the FTC's (in the U.S.) or ISO'ssmoking-machine methods which determine, for example, the amount of tarand nicotine generated when a cigarette is smoked by a machine inaccordance with standardized conditions. See Pillsbury et al., J. Assoc.Off. Analytical Chem. (1969); ISO: 4387 (1991). Most commercialcigarettes generally yield about 10 to 15 parts “tar” to every 1 partnicotine, measured in milligrams, as analyzed in PCT application WO2005/018307. Many public health officials believe that the currentFTC/ISO machine smoking regime is flawed since these methodologies failto take into account human smoking behavior which is primarily driven bynicotine seeking. In other words, these methods don't considercompensatory smoking. Compensatory smoking or compensation, as it isalso called, essentially means over smoking (smoking more intensively)due to the reduced presence of nicotine in tobacco smoke or undersmoking (smoking less intensively) due to the increased presence ofnicotine. See Benowitz, N. Compensatory Smoking of Low Yield Cigarettes,NCI Monograph 13.

Novel smoking-machine methods are currently being evaluated, especiallythose that consider compensatory smoking of low-yield brands. An exampleis a method involving the adjustment of smoking parameters so thatbrands with lower ISO nicotine yields are machine smoked more intensely.Kozlowski and O'Connor Lancet 355: 2159 (2000). Other proposed methodsmeasure yields of toxins on a per nicotine unit basis or at a defined“target” nicotine yield. This is achieved, for example, bysystematically varying puff volume, puff interval, and blockage ofventilation holes until the target nicotine yield is reached. Cigarettescan then be rated on the effort required to get the target nicotineyield as well as on toxin delivery at that yield. Consumers wouldbenefit from these smoking-machine methods since comparisons of specifictoxins among different brands could be evaluated.

Studies have suggested that many smokers inhale just as much smoke withmost “light” and “ultra-light” cigarettes as full flavor cigarettes(Gori and Lynch, Regulatory Toxicology and Pharmacology 5:314-326).Smokers may compensate or smoke lower-yield cigarettes (per the FTC orISO method) more aggressively (than higher-yield cigarettes) in order toobtain their desired nicotine impact and mouth feel of smoke, which areimportant sensory properties. Rose, J. E. “The role of upper airwaystimulation in smoking,” in Nicotine Replacement: A Critical Evaluation,p. 95-106, 1988.

The manner in which a smoker may compensate include the frequency ofpuffs per cigarette and volume of smoke inhalation of such puffs,duration of the smoke inhalation being held before exhaling, number ofcigarettes smoked within a specified time period, and the percentage ofeach cigarette that is smoked (how far down the cigarette is smoked).

When the percentage of nicotine per unit of inhaled smoke volumeincreases, many smokers may compensate and inhale less smoke. Gori G.B., Virtually Safe Cigarettes. Reviving an opportunity once tragicallyrejected. IOS Press. Amsterdam, (2000). The higher the percentage ofnicotine in cigarette tobacco, the higher the percentage of nicotine incigarette smoke. More specifically, the higher the percentage ofnicotine in a cigarette's filler, the higher the percentage of nicotinein cigarette smoke. “Filler” means any smokable material that is rolledwithin a cigarette or cigarette-like device and includes (a) alltobaccos, including but not limited to reconstituted and expandedtobaccos, (b) any non-tobacco substitutes that may accompany (a); (c)tobacco casings, (d) other additives including flavorings that (a), (b)or (c) are supplemented with. A cigarette-like device is any devicespecifically intended to deliver nicotine through an alternative “smoke”aerosol formed by heating tobacco materials. Characteristics of suchdevices are that they contain a high content of glycerin or a glycerinsubstitute with minimal or no combustion pyrolysis. Glycerin whenheated, vaporizes rapidly and forms an aerosol that can be inhaled andis very similar in appearance and feel to conventional cigarette smoke.

Therefore, the nicotine content of tobacco filler contained within acigarette or cigarette-like device, all other factors held constant(including but not limited to, the type of filter, cigarette paperincluding its porosity, plug wraps, and tipping paper utilized, and theamount of filter ventilation), would roughly have to double for acorresponding two-fold increase of nicotine in mainstream cigarettesmoke. Further, the nicotine content of tobacco filler contained withina cigarette or cigarette-like device, all other factors held constant,would roughly have to triple for a corresponding three-fold increase ofnicotine in mainstream cigarette smoke. The calculations in this sectionrefer to protonated nicotine in the mainstream smoke of a cigaretteaugmented with the described increase in nicotine levels and not “free”or “volatile” nicotine.

In one preferred embodiment of the invention a reduced-exposurecigarette is manufactured comprising an increased-nicotine tobacco planthaving up to a two-fold increase of nicotine. In another preferredembodiment of the present invention a reduced-exposure cigarette ismanufactured comprising an increased-nicotine tobacco having greaterthan a two-fold increase of nicotine.

The variety testing program conducted through the Agricultural ResearchService at North Carolina State University evaluates breeding linesthrough the Regional Minimum Standards Program and commercial varietiesthrough the North Carolina Official Variety Test (NCOVT). The reportedtotal alkaloid concentration of flue-cured commercial varieties in theNCOVT, Three-Year Average from 2001-2003, range from 2.45 to 3.17percent. Smith et al., “Variety Information,” Chapter 3 in: NORTHCAROLINA OFFICIAL VARIETY TEST (2004), Table 3.1. The three-year averagetotal alkaloid concentration was 2.5 percent for K-326, the cultivarutilized in Examples 4-6 and 9-14. The Spectrometric method is used tomeasure total alkaloid content of tobacco cultivars in the above NCOVTand for purposes of measuring total alkaloid content of tobacco andincreased-nicotine plants of the present invention. USA/TechnicalAdvisory Group ISO/TC 126—Tobacco and tobacco products: ISO/DIS 2881. Asdisclosed herein including within the figures, it is apparent that someincreased-nicotine plants have the capacity to at least double or evenmore than triple the nicotine accumulation of the wild-type K326controls. Likewise, in Examples 4 to 6, NBB1 and A622 overexpressionproduces a two- or even a three-fold increase in nicotine compared withwild-type controls.

“Curing” is the aging process that reduces moisture and brings about thedestruction of chlorophyll giving tobacco leaves a golden color and bywhich starch is converted to sugar. Cured tobacco therefore has a higherreducing sugar content and a lower starch content compared to harvestedgreen leaf “Flue-cured tobacco” refers to a method of drying tobaccoplants in a ventilated barn with heat and is characterized by a uniquecolor, high reducing sugar content, medium to heavy in body andexceptionally smooth smoking properties. Bacon et al., Ind. Eng. Chem.44: 292 (1952).

Tobacco-specific nitrosamines (TSNAs) are a class of carcinogens thatare predominantly formed during curing and processing. Hoffman, D., etal., J. Natl. Cancer Inst. 58, 1841-4 (1977); Wiernik A et al., RecentAdv. Tob. Sci, (1995), 21: 39-80. TSNAs, such as4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), andN′-nitrosoanabasine (NAB), are formed by N-nitrosation of nicotine andother minor Nicotiana alkaloids, such as nornicotine, anatabine, andanabasine.

Increased-nicotine tobacco may contain higher nitrosamines since thereis a positive correlation between alkaloid content in tobacco and TSNAaccumulation. For example, a significant correlation coefficient betweenanatabine and NAT was found to be 0.76. Djordjevic et al., J. Agric.Food Chem., 37: 752-56 (1989). However, U.S. Pat. No. 5,803,081, U.S.Pat. No. 6,135,121, U.S. Pat. No. 6,805,134, U.S. Pat. No. 6,895,974 andU.S. Pat. No. 6,959,712 and U.S. Published Applications 2005/0034365,2005/0072047, 2005/0223442, 2006/0041949, and PCT published applicationWO 2006/091194, and others, discuss methodology to reducetobacco-specific nitrosamines, which can be applied to a tobacco productthat utilizes the subject invention.

Accordingly, the present invention provides constructs and methodologyfor producing cigarettes and other tobacco products containing increasednicotine levels. A desirable reduced-exposure cigarette should deliver asmoker's desired level of nicotine per cigarette as efficiently aspossible while maintaining acceptable taste. See WO 05/018307.

Reconstituted Tobacco, Expanded Tobacco and Blending

Increased-nicotine tobacco also may be used to produce reconstitutedsheet tobacco (Recon) and expanded tobacco or puffed tobacco. Recon canbe produced from the following: tobacco dust, stems, small leafparticles, other byproducts of tobacco processing and cigarettemanufacturing, and sometimes straight whole leaf. The recon process,which varies by manufacturer, closely resembles the typical paper makingprocess and entails processing the various tobacco fractions and thencutting the recon sheets into a size and shape that resembles cigarettetobacco (cut-rag tobacco).

In addition, increased-nicotine tobacco may be used, according to thepresent invention, to produce expanded tobacco, also known as puffedtobacco, which is an important component in many cigarette brands.Expanded tobacco is made through expansion of suitable gases, wherebythe tobacco is “puffed,” resulting in reduced density and greaterfilling capacity, which in turn reduces the weight of tobacco used incigarettes. By using increased-nicotine tobacco as a starting material,cigarettes made with the resultant expanded tobacco will yield reducedratios of toxic chemicals, such as tar and carbon monoxide, to nicotine.

Increased-nicotine expanded tobacco, increased-nicotine Recon, andincreased-nicotine cut-rag tobacco can be blended at any percentageamong the three or with any percentages of non-transformed expandedtobacco, non-transformed recon or non-transformed cut-rag to producecigarette filler containing varying nicotine contents. Any such blend isthen incorporated into the cigarette making process according tostandard methods known in the art.

Tobacco products other than cigarettes utilizing GE increased-nicotinetobacco are manufactured using any of the tobacco plant materialdescribed herein according to standard methods known in the art. In oneembodiment, tobacco products are manufactured comprising plant materialobtained from increased-nicotine tobacco. The increased-nicotine contentcan be up to greater than three times that of wild type cultivars.

Nicotinic Alkaloid Enzymes and Analogs

In addition to traditional tobacco products, such as cigarettes andchewing tobacco, the present invention provides methodology forproducing nicotine and nicotine analogs, as well as enzymes forsynthesis of nicotine and nicotine analogs. These compounds may beproduced by genetically engineered nicotine-producing plants andnon-nicotine producing cells, as well as in a cell-free/in vitro system.

Because recent studies suggest a role for nicotine receptors in treatinga variety of conditions and disorders, such as Alzheimer's disease,schizophrenia, central and autonomic nervous systems dysfunction, andaddictions, there is a need for nicotine receptor ligand sources. Forexample, the inventive methods and constructs may be used for producingnicotinic alkaloids. It has been shown that transgenic hairy rootcultures overexpressing PMT provide an effective means for large-scalecommercial production of scopolamine, a pharmaceutically importanttropane alkaloid. Zhang et al., Proc. Nat'l Acad. Sci. USA 101: 6786-91(2004). Accordingly, large-scale or commercial quantities of nicotinicalkaloids can be produced in tobacco hairy root culture by expressing atleast one of A622 and NBB1. Likewise, the present invention contemplatescell culture systems, such as bacterial or insect cell cultures, forproducing large-scale or commercial quantities of nicotine by expressingA622 and NBB1.

Additionally, products can be made directly using the activity of NBB1and A622 enzymes. For example, recombinant NBB1 and A622 enzymes may beused for the synthesis, or partial synthesis, of nicotinic alkaloids ornicotinic alkaloid analogs. Accordingly, large-scale or commercialquantities of A622 and NBB1 can be produced by a variety of methods,including extracting recombinant enzyme from a genetically engineeredplant, cell, or culture system, including but not limited to hairy rootcultures, insect, bacterial, fungal, plant, algae, and mammalian cellculture, or in vitro.

Specific examples are presented below of methods for identifyingsequences encoding enzymes involved in nicotine biosynthesis, as well asfor introducing the target gene to produce plant transformants. They aremeant to be exemplary and not as limitations on the present invention.

Example 1: Identification of NBB1 as a Gene Regulated by the NIC Loci

A cDNA microarray prepared from a Nicotiana sylvestris-derived cDNAlibrary, see Katoh et al., Proc. Japan Acad. 79, Ser. B: 151-54 (2003),was used to search for novel genes which are controlled by the nicotinebiosynthesis regulatory NIC loci.

N. sylvestris cDNAs were amplified by PCR and spotted onto mirror-coatedslides (type 7 star, Amersham) using an Amersham Lucidea array spotter.DNA was immobilized on the slide surface by UV crosslinking (120 mJ/m²).N. tabacum Burley 21 plantlets (WT and nic1nic2) were grown onhalf-strength B5 medium supplemented with 1.5% (W/V) sucrose and 0.35%(W/V) gellan gum (Wako) in Agripot containers (Kirin).

Roots of eight-week-old plantlets were harvested, immediately frozenwith liquid nitrogen, and kept at −80° C. until use. Total RNA wasisolated using Plant RNeasy Mini kit (Qiagen) from the frozen roots, andmRNA was purified using GenElute mRNA Miniprep kit (Sigma). cDNA wassynthesized from 0.4 μg of the purified mRNA by using LabelStar ArrayKit (Qiagen) in the presence of Cy3 or Cy5-dCTP (Amersham). cDNAhybridization to the microarray slides and post-hybridization washeswere performed using a Lucidea SlidePro hybridization machine(Amersham). Microarrays were scanned using an FLA-8000 scanner(Fujifilm). Acquired array images were quantified for signal intensitywith ArrayGauge software (Fujifilm). cDNA probes from wild type andnic1nic2 tobacco were labeled with Cy3 and Cy5 in reciprocal pair-wisecombinations. Hybridization signals were normalized by accounting forthe total signal intensity of dyes. cDNA clones which hybridized towild-type probes more than twice as strongly compared to nic1nic2 probeswere identified, and these included NBB1.

Full-length NBB1 cDNA was obtained by 5′- and 3′-RACE from total RNA ofN. tabacum by using a SMART RACE cDNA Amplification Kit (Clontech). Theresultant full-length NBB1 cDNA sequence was cloned into pGEM-T vector(Promega) to give pGEMT-NBB1cDNAfull.

The nucleotide sequence of the NBB1 cDNA insert was determined on bothstrands using an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems)and a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).The NBB1 full-length cDNA is set forth in SEQ ID NO: 1. The amino acidsequence encoded by the nucleotide sequence is set forth in SEQ ID NO:2. The protein sequence includes a FAD-binding motif. A putativevacuolar signal peptide is located at the N-terminus.

Example 2: Characterization of NBB1

NBB1 expression in tobacco plants was investigated by Northern blotanalysis.

Plants of Nicotiana tabacum cv. Burley 21 (abbreviated below as WT) andmutant lines in which nic1, nic2 or both nic1 and nic2 mutations hadbeen introduced in the Burley 21 background were grown in vitro for 2months at 25° C. with 150 μmole photons/m² of light (16 h light, 8 hdark) on ½×B5 medium with 3% sucrose and 0.3% gellan gum. The plantswere treated with methyl jasmonate vapor by adding 0.5 mL of 100 μMmethyl jasmonate to an Agripot container (Kirin, Tokyo) with a solidmedium capacity of 80 cm³ and a gas capacity of 250 cm³ containing theplants. The treatment times were set at 0 h and 24 h. The root parts andleaf parts (2^(nd) through 6^(th) leaves from a plant body with a totalof 7 to 10 leaves) were collected from the plant bodies and immediatelystored frozen using liquid nitrogen.

RNA was extracted using an RNeasy Midi Kit (Qiagen) according to themanufacturer's protocol. However, polyvinyl pyrrolidine was added to aconcentration of 1% to RLT Buffer in the Qiagen kit. The columnoperation was performed twice to increase the purity of the RNA.

RNA blotting was carried out according to the methods given by Sambrookand Russell (Sambrook, J. et al., Molecular Cloning, Cold Spring HarborLaboratory, Chapter 7 (2001)).

The sequence fragment from 1278 bp through the end (1759 bp) of the NBB1nucleotide sequence (SEQ ID NO: 1) was used as the probe template. Thetemplate was prepared by amplification from the cDNA clone by PCR usingthe following primers:

primer 1: GGAAAACTAACAACGGAATCTCT (SEQ ID NO: 13) primer 2:GATCAAGCTATTGCTTTCCCT (SEQ ID NO: 14)

The probe was labeled with ³²P using a Bcabest labeling kit (Takara)according to the manufacturer's instructions. Hybridization wasaccomplished using ULTRAhyb (Ambion) as the buffer according to themanufacturer's protocol.

PMT probe was prepared from a PMT sequence cloned into a pcDNAII vectorin E. coli (Hibi et al., 1994). The plasmid was extracted and purifiedfrom the E. coli using a QIAprep Spin Miniprep Kit (Qiagen), treatedwith the restriction enzymes XbaI and HindIII by ordinary methods, andthe digested DNA was electrophoresed through an agarose gel. DNAfragments of about 1.5 kb were collected using a QIAquick Gel ExtractionKit (Qiagen). The collected DNA fragments were ³²P labeled by the samemethods used for the NBB1 probe, and hybridized.

As FIG. 1 shows, NBB1 and PMT have the same pattern of expression intobacco plants. Evidence that NBB1 is involved in nicotine biosynthesisis that, like PMT, NBB1 is under the control of the NIC genes, and itexhibits a similar pattern of expression to PMT.

Example 3: Phylogenetic Analysis of NBB1

NBB1 polypeptide has 25% identity and 60% homology to the Eschscholziacalifornica berberine bridge enzyme (BBE). Dittrich et al., Proc. Nat'lAcad. Sci. USA 88: 9969-73 (1991)). An alignment of the NBB1 polypeptidewith EcBBE is shown in FIG. 2.

A phylogenetic tree was constructed using the sequences of NBB1polypeptide and plant BBE-like polypeptides, based on Carter &Thornburg, Plant Physiol. 134: 460-69 (2004). The phylogenetic analysiswas performed using neighbor-joining method with the CLUSTAL W program.Numbers indicate bootstrap values from 1,000 replicates. The sequencesused were: EcBBE, California poppy BBE (GenBank accession no. AF005655);PsBBE, opium poppy (Papaver somniferum) probable reticuline oxidase(AF025430); BsBBE, barberry (Berberis stolonifera) BBE (AF049347);VuCPRD2, cowpea (Vigna unguiculata) drought-induced protein (AB056448);NspNEC5, Nicotiana sp. Nectarin V (AF503441/AF503442); HaCHOX, sunflower(Helianthus annuus) carbohydrate oxidase (AF472609); LsCHOX, lettuce(Lactuca sativa) carbohydrate oxidase (AF472608); and 27 Arabidopsisgenes (At1g01980, At1g11770, At1g26380, At1g26390, At1g26400, At1g26410,At1g26420, At1g30700, At1g30710, At1g30720, At1g30730, At1g30740,At1g30760, At1g34575, At2g34790, At2g34810, At4g20800, At4g20820,At4g20830, At4g20840, At4g20860, At5g44360, At5g44380, At5g44390,At5g44400, At5g44410, and At5g44440).

The results are shown in FIG. 3. The three known BBEs form a separateclade and are underlined and indicated as “True BBEs.” The sequence ofNBB1 is not highly similar to any of the BBE or BBE-like proteins, andis separated from the other sequences at the base of the tree. The onlyother BBE-like protein described from the genus Nicotiana, nectarin V, aprotein described in nectar of the a hybrid ornamental Nicotianalangsdorffii×N. sanderae, Carter and Thornburg (2004), clusters with thecowpea drought-induced protein and several putative BBE-like proteinsfrom Arabidopsis. Because the nectar of the ornamental tobacco lacksalkaloids and nectarin V has glucose oxidase activity, it was concludedthat nectarin V is involved in antimicrobial defense in flowers and isnot likely to have any role in alkaloid synthesis. Id.

Example 4: NBB1 Overexpression in Tobacco Hairy Roots

Preparation of NBB1 Overexpression Construct

An attB-NBB1 fragment was amplified by PCR using the pGEMT-NBB1cDNAfullvector of Example 1 as the template and two sets of primers; one set forthe NBB1 gene-specific amplification (gene-specific primers) and anotherset to add the attB sequences (adapter primers). PCR conditions werethose recommended by the manufacturer. The GATEWAY entry clonepDONR221-NBB1 was created by a BP recombination reaction between theattB-NBB1 PCR product and pDONR221(Invitrogen).

Gene-Specific Primers

NBB1-attB1 (SEQ ID NO: 15) 5′ AAAAAGCAGGCTCACCATGTTTCCGCTCATAATTCTGNBB1-attB2 (SEQ ID NO: 16) 5′ AGAAAGCTGGGTTCATTCACTGCTATACTTGTGCAdapter Primers

attB1 adapter (SEQ ID NO: 17) 5′ GGGGACAAGTTTGTACAAAAAAGCAGGCT attB2adapter (SEQ ID NO: 18) 5′ GGGGACCACTTTGTACAAGAAAGCTGGGTDescription of pTobRD2-DEST

A TobRD2 promoter region (SEQ ID NO: 5 in WO9705261) of 1,031 bp wasamplified using Burley 21 genomic DNA as the template and the TobRD2promoter-specific primers, and then digested with HindIII and XbaI.

TobRD2 Promoter-Specific Primers:

TobRD2-01F (SEQ ID NO: 19) 5′ AAAGCTTGGAAACATATTCAATACATTGTAG HindIIIsite is underlined. TobRD2-02R (SEQ ID NO: 20)5′ TCTAGATTCTACTACTATTTTATAAGTG XbaI site is underlined.

The resultant fragment was cloned between the HindIII and XbaI sites ofpBI101H (supplied from Dr. Shuji Yokoi of NAIST; ref. Molecular Breeding4: 269-275, 1998) resulting in plasmid pTobRD2-BI101H. A GATEWAYcassette containing attR recombination sites flanking a ccdB gene and achloramphenicol-resistance gene was cloned between the XbaI and SacIsites of the pTobRD2-BI101H binary vector to produce the binary vectorpTobRD2-DEST, which has a T-DNA region containing an NPTII geneexpression cassette (Nos promoter-neomycin phosphotransferase II ORF-Nosterminator) and an HPT gene expression cassette (CaMV 35Spromoter-hygromycin phosphotransferase ORF-Nos terminator) as selectionmarkers flanking a TobRD2 promoter adjacent to a GATEWAY cassette. Adiagram of the T-DNA region of pTobRD2-DEST is shown in FIG. 4A.

The NBB1 ORF was transferred by an LR reaction from the pDONR221-NBB1vector to a GATEWAY binary vector pTobRD2-DEST, which was designed toexpress a cloned ORF under the TobRD2 promoter. A diagram of the T-DNAregion of the final expression vector, pTobRD2-NBB1 ox, is shown in FIG.4B.

Production of Transgenic Hairy Roots

The binary vector pTobRD2-NBB1ox was introduced to Agrobacteriumrhizogenes strain 15834 by electroporation. Nicotiana tabacum cv. K326wild-type plants were transformed by A. rhizogenes using a leaf-discmethod, basically as described by Kanegae et al., Plant Physiol.105:2:483-90 (1994). Kanamycin resistance (200 mg/L in B5 medium) wasused as a selection marker for the pTobRD2-NBB1ox transformed lines (TNlines). Wild-type A. rhizogenes was used to produce control hairy rootlines (WT lines). Tobacco hairy roots were grown in the B5 liquid mediumat 27° C. under the dark condition for two weeks, and then harvested.

Procedure for Analyzing Expression

Expression levels of the NBB1 protein were analyzed by an immunoblotanalysis. Hairy roots were frozen in liquid nitrogen, and immediatelyhomogenized using a mortar and pestle in an extraction buffer (100 mMTris-HCl pH6.8, 4% SDS, 20% glycerol) containing 1 mMphenylmethylsulfonyl fluoride and 200 mM dithiothreitol. Aftercentrifugation of the homogenates, soluble proteins in the supernatantwere separated by SDS-PAGE. Immunoblot analysis was performed using ananti-NBB1 rabbit serum. The detailed procedures were reportedpreviously. Shoji et al., Plant Mol. Biol., 50, 427-440 (2002).Immunoblots with anti-NBB1 antiserum show that the transgenic hairy rootlines TN9 and TN17 have increased levels of NBB1 protein. See FIG. 5A.

Procedure for Analyzing Alkaloid Levels

Transgenic hairy roots were cultured for two weeks, collected, andfreeze dried. 2 ml of 0.1 N. sulfuric acid was added to 19 mg of thefreeze-dried sample. This suspension was sonicated for 15 minutes, andfiltered. Ammonium hydroxide (0.1 ml, 28% NH₃; Wako) was added to 1 mlof the filtrate, and the mixture was centrifuged for 10 minutes at15,000 rpm. A sample of the supernatant (1 ml) was loaded onto anExtrelut-1 column (Merck) and let sit for 5 minutes. Alkaloids wereeluted with 6 ml of chloroform. The eluted chloroform fraction was thendried under reduced pressure at 37° C. with an evaporator (TaitecConcentrator TC-8). The dried sample was dissolved in 50 μl of ethanolsolution containing 0.1% dodecane. A gas chromatography apparatus(GC-14B, Shimadzu) equipped with a capillary column (Rtx-5Amine column,Restec) and an FID detector was used to analyze the samples. The columntemperature was maintained at 100° C. for 10 min, elevated to 150° C. at25° C./min, held at 150° C. for 1 min, elevated to 170° C. at 1° C./min,held at 170° C. for 2 min, elevated to 300° C. at 30° C./min, and thenheld at 300° C. for 10 min. Injection and detector temperature was 300°C. A 1 μl sample of the purified alkaloid preparation was injected, andalkaloid contents were measured by the internal standard method.

The hairy root lines transformed with the NBB1 overexpression vectorpTobRD2-NBB1ox (TN9, TN17) have increased levels of nicotine andnornicotine compared to wild type hairy root lines. See FIG. 6.

Example 5: A622 Overexpression in Tobacco Hairy Roots

Preparation of A622 Overexpression Construct

An attB-A622 fragment was amplified using the pcDNAII-A622 vector, perHibi et al, Plant Cell 6: 723-35 (1994), as the template, theA622-specific primers below, and adapter primers, as described above forExample 4.

Gene-Specific Primers

A622-attB1 (SEQ ID NO: 21)5′ AAAAAGCAGGCTTCGAAGGAGATAGAACCATGGTTGTATCAGAGAAA AGCA A622-attB2 (SEQID NO: 22) 5′ AGAAAGCTGGGTCCTAGACAAATTTGTTGTAGAACTCGTCG

The amplified A622 fragment was cloned into the pDONR221 vector by BPreaction, resulting in pDONR-A622, and then the A622 fragment wastransferred from pDONR-A622 to pTobRD2-DEST by an LR reaction. Theresultant expression vector was referred to as pTobRD2-A622ox. A diagramof the T-DNA region of pTobRD2-A622ox is shown in FIG. 4C.

Production of Transgenic Hairy Roots

N. tabacum cv. K326 wild-type plants were transformed by A. rhizogenes15834 containing the pTobRD2-A622ox vector, as described above forExample 4. Transgenic hairy roots carrying the T-DNA from pTobRD2-A622oxwere referred to as TA lines, and cultured as described above in Example4.

Procedure for Analyzing Expression

Immunoblot analysis was performed as described above in Example 4,except that anti-A622 mouse serum was used for A622 protein detection.Hairy root lines TA11, TA14, TA21 transformed with the A622overexpression vector have higher levels of A622 protein. See FIG. 5B

Procedure for Analysis of Alkaloid Levels

Tobacco alkaloids were extracted, purified, and analyzed as describedabove in Example 4. Hairy root lines TA11, TA14, TA21 transformed withthe A622 overexpression vector have higher levels of nicotine andnornicotine. See FIG. 6.

Example 6: NBB1 and A622 Overexpression in Tobacco Hairy Roots

Preparation of A622 and NBB1 Overexpression Construct

In order to express both A622 and NBB1 proteins from a single T-DNA, theTobRD2-A622 expression cassette and the TobRD2-NBB1 expression cassettewere cloned in tandem in a binary vector. The TobRD2-A622 cassette wascut from pTobRD2-A622ox with HindIII, and then cloned into the HindIIIsite at the 5′ end of the TobRD2 promoter in pTobRD2-NBB1ox. Theresultant vector for overexpression of both NBB1 and A622 was referredto as pTobRD2-A622ox-NBB1ox. A diagram of the T-DNA region ofpTobRD2-A622ox-NBB1ox is shown in FIG. 4D.

Production of Transgenic Hairy Roots

Nicotiana tabacum cv. K326 wild-type plants were transformed by A.rhizogenes 15834 containing the pTobRD2-A622ox-NBB1 ox vector, asdescribed above for Example 4. Transgenic hairy roots carrying the T-DNAfrom pTobRD2-A622ox-NBB1ox were referred to as TNA lines, and culturedas described above in Example 4.

Procedure for Analyzing Expression

Immunoblot analysis was performed as described above in Example 4,except that both anti-A622 mouse serum and anti-NBB1 rabbit serum wereused for protein detection. In the hairy root line TNA8 the expressionlevel of both NBB1 (see FIG. 5A) and A622 (see FIG. 5B) are increasedcompared to wild type hairy root lines.

Procedure for Analyzing Alkaloid Levels

Tobacco alkaloids were extracted from hairy root line TNA8, purified,and analyzed as described above in Example 4. The levels of nicotine andnornicotine are higher in line TNA8 than in wild type hairy root lines.(See FIG. 6)

Example 7: Transgenic A. belladonna Plants Expressing A622 Protein

Atropa belladonna produces tropane alkaloids, hyoscyamine andscopolamine, which are derived from the N-methylpyrrolinium cation, butdoes not contain nicotine alkaloids, possibly due to the absence of NBB1and A622 genes.

Tobacco A622 cDNA containing an introduced NcoI site at the first ATGwas excised from the pcDNAII-A622 vector (Hibi et al. 1994) as anNcoI-BamHI fragment and cloned into pRTL2 (Restrepo et al., Plant Cell2:987-98 (1990)) under the control of a CaMV35S promoter with aduplicated enhancer. This A622 overexpression cassette was excised withHindIII and cloned in a binary vector pGA482 (Amersham) to produce theA622 expression vector pGA-A622.

Production of Transgenic Plants

The binary vector pGA-A622 was introduced to A. tumefaciens strainEHA105 by electroporation. A. belladonna plants were transformed by A.tumefaciens using a leaf-disk method, basically as described by Kanegaeet al., (Plant Physiol. 105(2):483-90 (1994)). Kanamycin resistance (200mg/L in MS/B5 medium) was used as a selection marker for the pGA-A622transformation. Transgenic 35S-A622 plants were regenerated from theleaf discs, grown at 22° C. under continuous light in a growth chamber.

Procedure for Analyzing Expression

Total proteins were extracted from leaves of wild-type and 35S-A622 T1plants, as described above in Example 5 Immunoblot analysis wasperformed using anti-A622 mouse serum. Leaf tissues of theself-pollinated T1 generation plants that contained high amounts of A622protein, such as line C1#3 (see FIG. 7), were used for alkaloidanalysis.

Procedure for Analysis of Alkaloid Levels

Nicotinic alkaloids in transgenic A. belladonna plants were extractedwith 1M H₂SO₄ and purified basically as described (Hashimoto et al.,1992). Alkaloids were identified by gas chromatography-mass spectrometry(GC-MS) (Hewlett Packard 5890 series II/JEOL MStation JMS700 with HP-5mscolumn) after comparison of their mass spectra to those of authenticstandards. The column temperature was maintained at 100° C. for 10 min,elevated to 150° C. at 25° C./min, held at 150° C. for 1 min, elevatedto 170° C. at 1° C./min, held at 170° C. for 2 min, elevated to 300° C.at 30° C./min, and then held at 300° C. for 10 min. Introduction of A622alone in A. belladonna did not result in accumulation of nicotine orother nicotine alkaloids.

Example 8: Transgenic A. belladonna Hairy Roots Expressing NBB1 Proteinand A622 Protein

Preparation of NBB1 Overexpression Construct

To test whether the combination of NBB1 and A622 is sufficient for thecoupling reaction of nicotinic alkaloids, we produced transgenic A.belladonna hairy roots that express both A622 and NBB1, by transformingleaves of the A622-expressing Atropa plants of Example 7 with A.rhizogenes strain 15834, which carries an NBB1 expression vector.

Description of pBI101H-E2113-DEST

The binary vector pBE2113 carrying CaMV35S promoter with a duplicatedenhancer (E12) and 5′-upstream sequence of tobacco mosaic virus (Ω) wasobtained from Dr. Yuko Ohashi, National Institute of AgrobiologicalResources (Tsukuba, Japan), see Plant Cell Physiol. 37: 49-59 (1996),and was converted into a GATEWAY destination vector after the GATEWAYcassette, containing attR recombination sites flanking a ccdB gene and achloramphenicol-resistance gene, was cloned between the XbaI and SacIsites in the vector, which replaced the β-glucronidase gene with theGATEWAY cassette. The resultant destination binary vector was digestedwith HindIII and SacI, and the HindIII-SacI fragment containing theE12-35S-Ω-GATEWAY cassette was cloned between the HindIII and SacI sitesin pBI101H. The resultant destination binary vector was referred to aspBI101H-E2113-DEST. A diagram of the T-DNA region of pBI101H-E2113-DESTis shown in FIG. 4E.

NBB1 ORF was transferred from the pDONR221-NBB1-2 vector to the GATEWAYbinary vector pBI101H-E2113-DEST by an LR reaction. The expressionvector was referred to as pE1235SΩ-NBB1. A diagram of the T-DNA regionof pE1235SΩ-NBB1 is shown in FIG. 4F.

Production of Transgenic Hairy Roots

The binary vector pE1235SΩ-NBB1 was introduced to A. rhizogenes strain15834 by electroporation. Leaf tissues of the T1 generation plantcontaining a 35S-A622 cassette that expresses A662 (line C1#3) weretransformed with A. rhizogenes carrying pE1235SΩ-NBB1 using a leaf-discmethod, basically as described by Kanegae et al. (Plant Physiol.105(2):483-90 (1994)). Hygromycine resistance (30 mg/L in B5 medium) wasused as a selection marker. Transgenic hairy roots carrying the T-DNAfrom pE1235SΩ-NBB1 (line E) and transgenic hairy root infected wild-typeA. rhizogenes without T-DNA as the control (WT line) were grown in theMS/B5 liquid medium for two weeks and then harvested.

Expression Analysis

Total proteins were extracted and immunoblot analysis was performed asdescribed above in Example 4. Anti-A622 mouse serum was used for A622protein detection, whereas anti-NBB1 rabbit serum was used for NBB1protein detection. A transgenic A. belladonna hairy root line (E2)expresses high amounts of both NBB1 and A622 proteins (see FIG. 8).

Alkaloid Analysis

The transgenic A. belladonna E2 and WT hairy roots were cultured for 3weeks in 100 ml of MS/B5 liquid medium containing 100 mg/l nicotinicacid. Nicotine alkaloids in E2 and WT hairy roots were extracted with 1MH₂SO₄ and purified basically as described (Hashimoto et al., 1992).Alkaloids were identified by gas chromatography-mass spectrometry(GC-MS) (Hewlett Packard 5890 series II/JEOL MStation JMS700 with HP-5mscolumn) after comparison of their mass spectra to those of authenticstandards. The column temperature was maintained at 100° C. for 10 min,elevated to 150° C. at 25° C./minute, held at 150° C. for 1 minute,elevated to 170° C. at 1° C./min, held at 170° C. for 2 minutes,elevated to 300° C. at 30° C./min, and then held at 300° C. for 10minutes.

A small but distinct novel peak was detected (See peak 5 in FIG. 9). Apeak corresponding to the peak 5 was not detectable in the WT line hairyroots. The compound of peak 5 showed an MS fragmentation profileidentical to that of nicotine, as shown in FIG. 10. This demonstratedthat expression of exogenous NBB1 and A622 are sufficient for nicotineformation in A. belladonna hairy roots.

Example 9: Increasing Nicotine Content by Expression of PMT UnderControl of the A622 Promoter

Description of pA622pro-DEST

pA622pro-DEST has the NPTII gene expression cassette and the HPT geneexpression cassette as selection markers. An A622 promoter of 1,407 bpwas amplified using a vector containing the A622 promoter (Shoji et al.,Plant Mol. Biol. 50, 427-440 (2002)) as the template and the A622promoter-specific primers shown below, and digested with HindIII andXbaI. The resultant fragment was cloned between the HindIII and XbaIsites in pBI101H. The binary vector was converted into a GATEWAYdestination vector after the GATEWAY cassette containing attRrecombination sites flanking a ccdB gene and achloramphenicol-resistance gene were cloned between the XbaI and SacIsites in the binary vector. See FIG. 11A.

A622 Promoter-Specific Primers

A622pro-01F (SEQ ID NO: 23) 5′ AAAAGCTTAGATCTCTCTTATGTTTCATG HindIIIsite is underlined. A622pro-02R (SEQ ID NO: 24)5′ TCTAGATTTACTCCTAGGGGAAGAAAAAAAGTAGC XbaI site is underlined.Preparation of PMT Overexpression Construct

An attB-PMT fragment was amplified using the tobacco PMT vector in whichPMT ORF (NCBI accession number; D28506) was cloned in the BstXI site ofpcDNAII (Invitrogen) (See SEQ ID NO: 12) as the template, thegene-specific primers below, and attB sequence adapter primers, asdescribed above in Example 4. A GATEWAY entry clone pDONR221-PMT wascreated by a BP recombination reaction between the attB-PMT PCR productand pDONR221 (Invitrogen).

Gene-Specific Primers

PMT-attB1 (SEQ ID NO: 25) 5′ AAAAAGCAGGCTCAAAAATGGAAGTCATATC PMT-attB2(SEQ ID NO: 26) 5′ AGAAAGCTGGGTTTAAGACTCGATCATACTTC

The PMT ORF was transferred from the pDONR221-PMT vector to the GATEWAYbinary vector pA622pro-DEST by an LR reaction. The gene expressionvector was referred to as pA622pro-PMTox. See FIG. 11B.

Production of Transgenic Tobacco Plants

pA622pro-PMTox was transformed into Agrobacterium tumefaciens strainEHA105, which was used to transform wild-type K326 leaves. Transgenic T0shoots were regenerated, and were transferred to the rooting medium.Several rooted transgenic plants were transferred to soil.

Alkaloid Analysis

Alkaloids were extracted from the transgenic tobacco leaves andanalyzed, as described above in Example 4. The nicotine content inleaves of plants sampled 36 days after transfer to soil was analyzed.Several transgenic lines transformed with pA622pro-PMTox showed greaternicotine accumulation than the control lines, in which wild-type K326plants were transformed with the AG-GUS cassette. See FIG. 12.

Example 10: Increasing Nicotine Content by Expression of PMT UnderControl of the TobRD2 Promoter

Preparation of PMT Overexpression Construct

The PMT ORF was transferred from the pDONR221-PMT vector to the GATEWAYbinary vector pTobRD2-DEST (see FIG. 4A) by an LR reaction. The geneexpression vector was referred to as pTobRD2-PMTox. See FIG. 11C.

Production of Transgenic Tobacco Plants

pTobRD2-PMTox was transformed into Agrobacterium tumefaciens strainEHA105, which was used to transform wild-type K326 leaves. Transgenic T0shoots were regenerated, and were transferred to the rooting medium.Several rooted transgenic plants were transferred to soil.

Alkaloid Analysis

Alkaloids were extracted from the transgenic tobacco leaves andanalyzed, as described above in Example 4. The nicotine content inleaves of plants sampled 36 days after transfer to soil was analyzed.Several transgenic lines showed greater nicotine accumulation than thecontrol lines, in which wild-type K326 plants were transformed with aTobRD2-GUS cassette. See FIG. 13.

Example 11: Increasing Nicotine Content by Expression of QPT UnderControl of the A622 Promoter

Preparation of QPT Overexpression Construct

The QPT ORF fragment (SEQ ID NO: 11) was amplified using the pBJY6vector (supplied from Dr. Kenzo Nakamura, Nagoya University, Japan) asthe template and the gene-specific primers shown below. A GATEWAY entryclone pENTR-QPT was created by a TOPO cloning reaction.

QPT Gene-Specific Primers

QPT-F 5′ CACCATGTTTAGAGCTATTCC (SEQ ID NO: 27) QPT-R5′ TCATGCTCGTTTTGTACGCC (SEQ ID NO: 28)

The QPT ORF was transferred from the pENTR-QPT vector to the GATEWAYbinary vector pA622pro-DEST (see FIG. 11A) by an LR reaction. The geneexpression vector was referred to as pA622pro-QPTox. See FIG. 11D.

Production of Transgenic Tobacco Plants

pA622pro-QPTox was transformed into Agrobacterium tumefaciens strainEHA105, which was used to transform wild-type K326 leaves. Transgenic T0shoots were regenerated, and were transferred to the rooting medium.Several rooted transgenic plants were transferred to soil.

Analyzing Alkaloids

Alkaloids were extracted from the transgenic tobacco leaves andanalyzed, as described above in Example 4. The nicotine content inleaves of plants sampled 36 days after transfer to soil was analyzed.Several transgenic lines showed greater nicotine accumulation than thecontrol lines, in which wild-type K326 plants were transformed with anA622-GUS cassette. See FIG. 14.

Example 12: Increasing Nicotine Content by Expression of QPT UnderControl of the TobRD2 Promoter

Preparation of QPT Overexpression Construct

The QPT ORF was transferred from the pDONR221-QPT vector to a GATEWAYbinary vector pTobRD2-DEST (see FIG. 4A) by an LR reaction. The geneexpression vector was referred to as pTobRD2-QPTox. See FIG. 11E.

Production of Transgenic Tobacco Plants

pTobRD2-QPTox was transformed into Agrobacterium tumefaciens strainEHA105, which was used to transform wild-type K326 leaves. Transgenic T0shoots were regenerated, and were transferred to the rooting medium.Several rooted transgenic plants were transferred to soil.

Analyzing Alkaloids

Alkaloids were extracted from the transgenic tobacco leaves andanalyzed, as described above in Example 4. The nicotine content inleaves of plants sampled 36 days after transfer to soil was analyzed.Several transgenic lines showed greater nicotine accumulation than thecontrol lines, in which wild-type K326 plants were transformed with aTobRD2-GUS cassette. See FIG. 15.

Example 13: Increasing Nicotine Content by Expression of PMT and QPTUnder Control of the A622 Promoter

Description of pBI221-A622pro-DEST

pBI221-A622pro-DEST was the basic vector for construction of multi-geneexpression binary vector. An A622 promoter of 1,407 bp was amplifiedusing the pUC19-A622profull-LUC vector as the template and the A622promoter-specific primers, and digested with HindIII and XbaI. Theresultant fragment was cloned between HindIII and XbaI sites in pBI221(Clontech), which replaced the CaMV 35S promoter with the A622 promoter.The vector was converted into a GATEWAY destination vector after theGATEWAY cassette containing attR recombination sites flanking a ccdBgene and a chloramphenicol-resistance gene was cloned between the XbaIand SacI sites in the vector, which replaced the B-glucronidase genewith the GATEWAY cassette. Then, an HindIII-EcoRI adapter was insertedinto the EcoRI site at the 3′ end of Nos terminator resulting inpBI221-A622pro-DEST.

Preparation of PMT-QPT Overexpression Construct

In order to overexpress both PMT and QPT proteins, the A622pro-PMTexpression cassette and the A622pro-QPT expression cassette were clonedin tandem in a binary vector. First, the PMT ORF was transferred fromthe pDONR221-PMT vector to the GATEWAY binary vector pBI221-A622pro-DESTby an LR reaction. The gene expression vector was referred to aspBI221-A622pro-PMT. The pBI221-A622pro-PMT was digested with HindIII,and then cloned into the HindIII site at the 5′ end of A622 promoter ofthe pA622pro-QPTox vector. The resultant PMT-QPT expression vector wasreferred to as pA622pro-PMTox-QPTox. A diagram of the T-DNA region ofpA622pro-PMTox-QPTox is shown in FIG. 11F.

Production of Transgenic Tobacco Plants

pA622pro-PMTox-QPTox was transformed into Agrobacterium tumefaciensstrain EHA105, which was used to transform wild-type K326 leaves.Transgenic T0 shoots were regenerated, and were transferred to therooting medium. Several rooted transgenic plants were transferred tosoil.

Procedure for Analysis of Alkaloid Levels

Alkaloids were extracted from the transgenic tobacco leaves andanalyzed, as described above in Example 4. The nicotine content inleaves of plants sampled 36 days after transfer to soil was analyzed.Several lines transformed with A622pro-PMTox-QPTox showed greaternicotine accumulation than the control lines, in which wild-type K326plants were transformed with an A622-GUS cassette. See FIG. 16.

Example 14: Increasing Nicotine Content by Expression of PMT and QPTUnder Control of the TobRD2 Promoter

Preparation of PMT-QPT Overexpression Construct

In order to overexpress both PMT and QPT proteins under control of theTobRD2, the TobRD2-PMT expression cassette and the TobRD2-QPT expressioncassette were cloned in tandem in a binary vector. First, the PMT ORFwas transferred from the pDONR221-PMT vector to a GATEWAY binary vectorpBI221-TobRD2-DEST by an LR reaction. The gene expression vector wasreferred to as pBI221-TobRD2-PMT. The pBI221-TobRD2-PMT was digestedwith HindIII, and then cloned into the HindIII site at the 5′ end ofTobRD2 promoter in pTobRD2-QPTox. The resultant PMT-QPT expressionvector was referred to as pTobRD2-PMTox-QPTox. See FIG. 11G.

Production of Transgenic Tobacco Plants

pTobRD2-PMTox-QPTox was transformed into Agrobacterium tumefaciensstrain EHA105, which was used to transform wild-type K326 leaves.Transgenic T0 shoots were regenerated, and were transferred to therooting medium. Several rooted transgenic plants were transferred tosoil.

Procedure for Analysis of Alkaloid Levels

Alkaloids were extracted from the transgenic tobacco leaves andanalyzed, as described above in Example 4. The nicotine content inleaves of plants sampled 36 days after transfer to soil was analyzed.Several transgenic lines transformed with the TobRD2-PMTox-QPTox showedgreater nicotine accumulation than the control lines, wild-type K326plants transformed with a TobRD2-GUS cassette. See FIG. 17.

Example 15: Production of Nicotinic Alkaloids in Arabidopsis byExpression of NBB1 in Combination with Additional Alkaloid BiosyntheticEnzymes

Arabidopsis plants do not produce nicotinic alkaloids. However, aprecursor of a number of nicotinic alkaloids, nicotinic acid, is acommon metabolite. The effect of expressing both NBB1 and A622 togetherin Arabidopsis was tested. Because nicotine is an alkaloid of particularinterest, expression of PMT was included to increase the availability ofmethylputrescine, a precursor of the pyrrolidine ring in nicotine.

Preparation of A622 Overexpression Construct

Tobacco A622 cDNA, which contains an introduced NcoI site at the firstATG (Hibi et al., 1994), was excised from pcDNAII (Invitrogen) as anNcoI-BamHI fragment and cloned into pRTL2 (Restrepo et al., Plant Cell2:987-98 (1990)) under control of the CaMV35S promoter with a duplicatedenhancer. This A622 overexpression cassette was excised with HindIII andcloned in a binary vector pGA482 (Amersham) to produce the A622expression vector pGA-A622.

Production of Transgenic 35S-A622 Plants

The binary vector pGA-A622 was introduced to A. tumefaciens strainLBA4404 by electroporation. A. thaliana plants (ecotype: Wassilewskija(WS)) were transformed by A. tumefaciens using a callus induction-plantregeneration method, basically as described by Akama et al., Plant cellReports 12: 7-11 (1992). Kanamycin resistance (50 mg/L onShoot-Induction medium) was used as a selection marker for the pGA-A622transformation. Transgenic plants were regenerated from the callus,grown at 23° C. under 16 h light/8 h dark condition in a growth chamber.

Preparation of NBB1- and PMT-Overexpression Construct

NBB1 ORF (SEQ ID NO: 9) was transferred from the pDONR221-NBB1-2 vectorto a GATEWAY binary vector pGWB2 (see FIG. 18A) by LR reaction. The geneexpression vector, in which NBB1 is linked to the CAMV 35S promoter, isreferred to as p35S-NBB1. See FIG. 18B. Similarly, PMT ORF wastransferred from the pDONR221-PMT vector to the pGWB2 by LR reaction.The gene expression vector is referred to as p35S-PMT. See FIG. 18C.

Production of Transgenic 35S-A622-35S-NBB1 Plants, 35S-A622-35S-PMTPlants and 35S-A622-35S-NBB1-35S-PMT Plants

The binary vectors p35S-NBB1 and p35S-PMT were introduced to A.tumefaciens strain EHA105 by electroporation. T1 generation plantscarrying pGA-A622 were transformed by A. tumefaciens using a floral dipmethod, basically as described by Clough et al., Plant J. 16: 735-43(1998). Hygromycin resistance (25 mg/L on Shoot-Induction medium) wasused as a selection marker for the p35S-NBB1 and p35S-PMTtransformations. Transgenic plants were grown at 23° C. under 16 hlight/8 h dark condition in a growth chamber. Resultant transgenicplants were screened by genomic PCR using the 35S promoter primers andNBB1- or PMT-gene specific primers.

Primers for Screening the 35S-A622-35S-NBB1 Plants

35S-F (SEQ ID NO: 29) 5′ ACCCTTCCTCTATATAAGGAAG NBB1-l140 (SEQ ID NO:30) 5′TGAGCCCAAGCTGTTTCAGAATCCPrimers for Screening the 35S-A622-35S-PMT Plants

35S-F 5′ ACCCTTCCTCTATATAAGGAAG (SEQ ID NO: 31) PMT-01R5′ CGCTAAACTCTGAAAACCAGC (SEQ ID NO: 32)

The PCR positive 35S-A622-35S-NBB1 plants and 35S-A622-35S-PMT plantswere crossed to produce 35S-A622-35S-NBB1-35S-PMT plants. F1 progenywere screened by genomic PCR using each expression cassette specificprimer pair.

Total proteins were extracted from the PCR-positive lines. Frozen rootswere immediately homogenized in extraction buffer (100 mM Tris-HClpH6.8, 4% SDS, 20% glycerol) containing 1 mM phenylmethyl sulfonylfluoride and 200 mM dithiothreitol using mortar and pestle. Aftercentrifugation of the homogenates, soluble proteins in the supernatantwere separated by SDS-PAGE. Immunoblot analysis was performed usinganti-A622 mouse serum for A622 protein detection and anti-NBB1 rabbitserum for NBB1 protein detection as described in Shoji et al., PlantMol. Biol. 50: 427-40 (2002). Transgenic lines expression were obtainedthat contain both A622 and NBB1 polypeptides. See FIG. 19.

Procedure for Analyzing Alkaloid Levels

Transgenic lines expressing NBB1 and A622 were selected and used for thealkaloid analysis. Alkaloids were extracted from the transgenic tobaccoleaves and analyzed, as described above in Example 4. As shown in FIG.20, a new peak corresponding to the elution time of nicotine was foundin the NBB1-A622-PMT line but not in the A622-PMT line. This shows thatexpression of NBB1 and A622 together is more effective than expressionof A622 alone for production of nicotinic alkaloids in a plant that doesnot normally produce alkaloids.

Example 16: Expression of NBB1 and A622 in Non-Plant Cells

The Bac-to-Bac Expression System (Invitrogen) insect cell-baculovirusexpression system was used to express NBB1 and A622 proteins with 6×Histags (SEQ ID NO: 34) in insect cells. In order to make the expressionclone, NBB1 and A622 ORFs (SEQ ID NO: 9 and 10, respectively) weretransferred from respective DONR vectors (pDONR-NBB1-2, pDONR-A622) tothe GATEWAY vector pDEST10 (Invitrogen) by LR reactions. Resultantexpression clones were referred to as pDEST10-NBB1 and pDEST10-A622.

pDEST10-NBB1 and pDEST10-A622 were transformed into MAX EfficiencyDH10Bac Cells (Invitrogen), to recover the recombinant bacmid DNAs. PCRanalysis using a gene specific primer and an M13 reverse primer was usedto verify the presence of recombinant bacmids containing A622 and NBB1.See FIG. 21A.

Resultant recombinant bacmids containing respective gene expressioncassettes were transfected to the insect cell Sf9 with Cellfectin(Invitrogen). The Sf9 cells were infected with the virus stocks in tworounds to amplify and scale-up the virus.

NBB1 and A622 were produced in the insect cell cultures, as shown byimmunoblotting with anti-NBB1 and anti-A622 antisera. The recombinantproteins containing the 6×His tag were purified by adsorption on Ni-NTAcolumns followed by elution with 0.5 M imidazole. See FIG. 21B.

What is claimed is:
 1. A method for increasing nicotine in a Nicotianaplant, comprising overexpressing NBB1 in the plant, wherein NBB1 isoverexpressed by: a) providing a genetically engineered Nicotiana plantthat contains an expression construct comprising, in the 5′ to 3′direction, a promoter operably linked to a heterologous nucleic acidmolecule selected from the group consisting of: (i) the nucleotidesequence set forth in SEQ ID NO: 1; and (ii) a nucleotide sequence thatencodes a polypeptide having the amino acid sequence set forth in SEQ IDNO: 2; and (b) growing the genetically engineered Nicotiana plant underconditions whereby the expression construct increases levels of NBB1 inthe plant relative to a non-genetically engineered control plant grownunder similar conditions, wherein the genetically engineered Nicotianaplant has an increased nicotine content relative to the non-geneticallyengineered Nicotiana control plant.
 2. The method according to claim 1,further comprising overexpressing at least one of quinolatephosphoribosyl transferase (QPT), putrescine methyl transferase (PMT),and A622.
 3. The method according to claim 2, wherein QPT, PMT, A622,and a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 are overexpressed.
 4. An increased nicotine Nicotiana plant producedby the method according to claim 1, wherein the plant has an increasednicotine content relative to a non-genetically engineered control plant.5. An increased nicotine product produced from the increased nicotineNicotiana plant according to claim 4, wherein the product comprises theexpression construct.
 6. The product according to claim 5, wherein theproduct is selected from the group consisting of a cigarette, cigarettetobacco, a cigar, cigar tobacco, pipe tobacco, chewing tobacco, snuff,snus, leaf tobacco, shredded tobacco, cut tobacco, a pharmaceutical anda nutraceutical.
 7. A method for increasing nicotine and yield in aNicotiana plant, comprising: (a) crossing an increased nicotineNicotiana plant that expresses heterologous NBB1 with a high yieldingNicotiana plant; and (b) selecting progeny plant with increased nicotineand high yield, wherein the plant has an increased nicotine content andyield relative to a non-transformed control plant.
 8. The methodaccording to claim 7, wherein the increased nicotine Nicotiana plant isproduced by: (a) transforming a Nicotiana plant with a constructcomprising, in the 5′ to 3′ direction, a promoter operably linked to aheterologous nucleic acid molecule selected from the group consistingof: (i) the nucleotide sequence set forth in SEQ ID NO: 1; and (ii)nucleotide sequence that encodes a polypeptide having the amino acidsequence set forth in SEQ ID NO: 2; (b) regenerating transgenicNicotiana plants from the transformed Nicotiana plant; and (c) selectinga transgenic Nicotiana plant having increased-nicotine content relativeto a control plant, wherein the plant has an increased nicotine contentrelative to a non-transformed control plant.
 9. An increased nicotineand yield plant produced by the method of claim
 8. 10. A method forincreasing nicotine and yield in a Nicotiana plant, comprising: (a)transforming a Nicotiana plant with (i) a first construct comprising, inthe 5′ to 3′ direction, a promoter operably linked to a heterologousnucleic acid molecule selected from the group consisting of thenucleotide sequence set forth in SEQ ID NO: 1, and a nucleotide sequencethat encodes a polypeptide having the amino acid sequence set forth inSEQ ID NO: 2; and (ii) a second construct comprising, in the 5′ to 3′direction, a promoter operably linked to a heterologous nucleic acidencoding an enzyme that increases yield; (b) regenerating transgenicNicotiana plants from the transformed Nicotiana plant; and (c) selectinga transgenic Nicotiana plant having increased-nicotine content andincreased yield relative to a control plant, where in the plant has anincreased nicotine content and yield relative to a non-transformedcontrol plant.
 11. An increased nicotine and yield plant produced by themethod of claim
 10. 12. An increased nicotinic alkaloid product producedfrom the plant of claim 11, wherein the product is selected from thegroup consisting of a cigarette, cigarette tobacco, a cigar, cigartobacco, pipe tobacco, chewing tobacco, snuff, snus, leaf tobacco,shredded tobacco, cut tobacco, a pharmaceutical and a nutraceutical,wherein the product comprises the first and second constructs.
 13. Anincreased nicotinic alkaloid product produced from the plant of claim 9,wherein the product is selected from the group consisting of acigarette, cigarette tobacco, a cigar, cigar tobacco, pipe tobacco,chewing tobacco, snuff, snus, leaf tobacco, shredded tobacco, cuttobacco, a pharmaceutical and a nutraceutical, wherein the productcomprises the construct.
 14. A method for increasing nicotine in aNicotiana plant, comprising culturing the plant under conditionssuitable for growth, wherein the plant comprises an expression constructcomprising, in the 5′ to 3′ direction, a promoter operably linked to aheterologous nucleic acid molecule selected from the group consistingof: (a) the nucleotide sequence set forth in SEQ ID NO: 1; and (b) anucleotide sequence that encodes a polypeptide having the amino acidsequence set forth in SEQ ID NO: 2, wherein the plant overexpresses NBB1and has an increased nicotine content relative to a non-transformedNicotiana control plant.
 15. The method of claim 14, further comprisingoverexpressing at least one of quinolate phosphoribosyl transferase(QPT), putrescine methyl transferase (PMT), and A622.
 16. The method ofclaim 14, wherein QPT, PMT, A622, and a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 2 are overexpressed.
 17. Anincreased nicotine Nicotiana plant produced by the method according toclaim 14, wherein the plant has an increased nicotine content relativeto a non-genetically engineered control plant.
 18. An increased nicotineproduct produced from the increased Nicotiana plant according to claim17, wherein the product is selected from the group consisting of acigarette, cigarette tobacco, a cigar, cigar tobacco, pipe tobacco,chewing tobacco, snuff, snus, leaf tobacco, shredded tobacco, cuttobacco, a pharmaceutical and a nutraceutical, wherein the productcomprises the expression construct.